Non-synaptic exocytosis along the axon shaft and its regulation by the submembrane periodic skeleton
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (Review Commons)
Abstract
Neuronal communication relies on signaling molecules transferred via exo- and endocytosis throughout the brain. Historically, studies have focused on vesicle exo- and endocytosis and their release machinery at synapses, and much less is known about non-synaptic exocytosis. If and how vesicles can access the plasma membrane along the axonal shaft, overcoming the insulating layer of the membrane-associated periodic scaffold, remains unclear. Here, we used fast live-cell imaging of mature cultured hippocampal neurons expressing the vamp2-pHluorin reporter to map sponta-neous exocytosis along axons. We detected non-synaptic exocytic events along the axon shaft that concentrated at the axon initial segment. Perturbation of the membrane-associated actin-spectrin skeleton revealed its role in regulating shaft exocytosis, similarly to its recently demonstrated role in shaping axonal endocytosis. To visualize the nanoscale arrangement of exocytic locations, we developed a novel correlative live-cell/two - color, single-molecule localization microscopy (SMLM) approach. We observed that regions of exocytosis are devoid of the submembrane spectrin mesh, with these spectrin-free areas being spatially separated from the spectrin clearings that contain clathrin-coated pits. Overall, our work reveals a new process of spontaneous exocytosis along the axon shaft, and how the axonal submem-brane skeleton shapes a heterogeneous landscape that uniquely segregates vesicular trafficking events.
Article activity feed
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
The authors do not wish to provide a response at this time.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Wiesner et al. use a combination of state-of-the-art imaging techniques to visualize the exocytosis of vesicles labeled with vamp2-phluorin. This work builds on previous findings of the group and aims to quantify vesicle exocytosis along the axon and relate it to the location of actin rings (MPS). Exocytosis indeed occurs in axonal and dendritic regions ; however, at a significantly lower rate than in presynaptic terminals. Exceptionally, the AIS shows a remarkably high exocytosis rate compared to other axonal regions. Perturbation of the MPS with swinholide increases the nonsynaptic release of vamp2-phluorin. The spots supporting …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Wiesner et al. use a combination of state-of-the-art imaging techniques to visualize the exocytosis of vesicles labeled with vamp2-phluorin. This work builds on previous findings of the group and aims to quantify vesicle exocytosis along the axon and relate it to the location of actin rings (MPS). Exocytosis indeed occurs in axonal and dendritic regions ; however, at a significantly lower rate than in presynaptic terminals. Exceptionally, the AIS shows a remarkably high exocytosis rate compared to other axonal regions. Perturbation of the MPS with swinholide increases the nonsynaptic release of vamp2-phluorin. The spots supporting exocytosis along the axon lack spectrin but are spatially segregated from regions used for CCP formation.
This work takes advantage of last-generation optical microscopy approaches to provide a quantitative analysis of exocytosis along the axon in nonsynaptic regions. Findings are solid, and the segregation of spots supporting exocytosis and endocytosis is intriguing. However, it is unclear to me whether the results obtained reflect a general mechanism or if they are biased by the experimental approach. Specifically, I have these major comments:
Use of the term "spontaneous." I understand that the term "spontaneous" refers to exocytosis that "just" occurs. But exocytosis cannot be evaluated without considering electrical activity. Vamp2-phluorin has been extensively used to investigate neurotransmitter release. Since spontaneous neurotransmitter release occurs in the absence of action potentials, it is important to know how the rates of exocytosis are affected after incubation with TTX. These experiments are necessary to show if vesicles are indeed released spontaneously or if they require the presence of action potentials.
The rates of spontaneous exocytosis are expressed in μm² /hour because these events are quite infrequent. According to the methods section, typical recording times are 5 minutes or less (lines 522-533). It would be more appropriate to express values per minute to establish comparisons with other works. The goal is to understand what sort of vesicles are being exocytosed. This is a key question that must be addressed before exploring other aspects such as the relationship to spectrin or endocytosis. If the authors can provide more information about the types of vesicles being exocytosed, this work becomes very relevant. Since I am aware of the technical difficulties associated with this, some suggestions are: use a vamp2-apex2-phluorin construct and confirm vesicle identity by EM, or, use iGluSnFR to confirm neurotransmitter release along axons.
Minor comments:
- Since culture conditions promote synapse formation, could spontaneous exocytosis found along axons related to synapse formation? This aspect could be tested by co-staining with PSD-95 after fixation.
Significance
Significance:
This is state-of-the-art study of the cell biology of neurons. The works demonstrates that vesicles are exocytosed along the axon and describes the molecular characteristics of the cytoskeletal elements involved.
General assessment:
The main strength of the study is the quality and diversity of imaging approaches used. The main limitation is defining the type of vesicle being exocytosed. It is important to know if vesicles imaged contain neurotransmitters.
Advance:
This paper is technically sound and provides interesting new concepts about how exocytosis occurs in nonsynaptic regions.
Audience:
This paper is appropriate for an audience familiarized with cell biology or cellular and molecular neuroscience
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The manuscript by Wiesner et al examines the non-synaptic exocytosis of vesicles in axon initial segment (AIS) as well as proximal and distal axons. Using VAMP2-pHluorin authors convincingly demonstrated that exocytosis occurs in AIS, along the axon shaft, cell body and dendrite. Upon perturbing membrane-associated periodic skeleton (MPS) pharmacologically, exocytic events at the AIS seem to increase, suggesting a potential inhibition of exocytosis at the baseline by MPS. To test whether exocytosis occurs in the area where the MPS is disorganized, authors developed a novel correlative live-cell/super resolution microscopy where …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The manuscript by Wiesner et al examines the non-synaptic exocytosis of vesicles in axon initial segment (AIS) as well as proximal and distal axons. Using VAMP2-pHluorin authors convincingly demonstrated that exocytosis occurs in AIS, along the axon shaft, cell body and dendrite. Upon perturbing membrane-associated periodic skeleton (MPS) pharmacologically, exocytic events at the AIS seem to increase, suggesting a potential inhibition of exocytosis at the baseline by MPS. To test whether exocytosis occurs in the area where the MPS is disorganized, authors developed a novel correlative live-cell/super resolution microscopy where exocytic events are identified live by HiLo imaging, followed by fixing the cells and imaging spectrins using SMLM platform and identified the repetitive spectrin map with SReD detector. Using this approach, they have identified that exocytosis in proximal and distal axons occurs at the membrane area with less spectrin. This area is distinct from the clathrin-enriched area where the group has previously identified as the endocytic sites. The strength of the paper lies within the imaging techniques. However, for publication, the following concerns should be addressed.
Major concerns
The absence of spectrin mesh. Unlike their previous paper using platinum replica EM where filaments are clearly visible, they are using the antibodies against one end of the spectrin, and therefore, they can visualize the periodic distribution of spectrin ends but cannot visualize its meshwork in this study. In addition to this limitation, at thicker processes, molecules below and above the focal plane may or may not be visible, potentially creating the spectrin-less area at the center. Thus, the conclusion regarding the absence of spectrin meshes at the exocytic sites is not well supported.
The rate of exocytosis among controls. The rate of exocytosis at the AIS does not match between Fig. 2C and 3B (1.08 vs 0.64 events/um2/hour). Although the increase in the rate by swinA is relative to the DMSO control, the rate in swinA-treated neurons can be said similar to the control in Fig. 2C. So it is equally likely that DMSO is affecting the rate, rather than swinA. They need an additional control group with no treatment.
Alignment of pHluorin with SMLM images. Since the interpretation depends highly on the perfect alignment of live-cell images with SMLM data and fixation can alter the ultrastructure [PMC7339343], using internal structures like mitochondria as fiducials would be more helpful. However, adding discussion would suffice.
Minor concerns
- The data presented here do not support the claim that actin perturbation favors non synaptic exocytosis (line 205). Please revise the sentence.
Significance
General assessment: The strength of the paper is in the imaging techniques. Visualizing the exocytic sites along the axons relative to the MPS is novel. The limitation of the paper is the lack of an approach to fully visualize spectrin networks.
Advance: If they can provide more convincing data demonstrating that exocytic sites are devoid of the spectrin meshwork, this paper will establish a novel concept regarding how non-synaptic exocytosis occurs along the axon.
Audience: Researchers working in the neuronal cell biology field will be the main audience of the manuscript.
Reviewer's expertise: Neuronal cell biology, exocytosis and endocytosis, and imaging
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
This manuscript investigates non-synaptic exocytosis along the axon shaft and examines how the submembrane actin-spectrin skeleton shapes the distribution of exocytic sites. Using cultured hippocampal neurons expressing VAMP2-pHluorin, the authors map spontaneous exocytosis along axons and apply a correlative live-cell/super-resolution imaging workflow to visualize the nanoscale organization of spectrin gaps relative to exocytic hotspots. They report that axonal shaft exocytosis is enriched at the AIS, that perturbing the actin-spectrin lattice alters shaft exocytosis, and that exocytic sites generally correspond to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
This manuscript investigates non-synaptic exocytosis along the axon shaft and examines how the submembrane actin-spectrin skeleton shapes the distribution of exocytic sites. Using cultured hippocampal neurons expressing VAMP2-pHluorin, the authors map spontaneous exocytosis along axons and apply a correlative live-cell/super-resolution imaging workflow to visualize the nanoscale organization of spectrin gaps relative to exocytic hotspots. They report that axonal shaft exocytosis is enriched at the AIS, that perturbing the actin-spectrin lattice alters shaft exocytosis, and that exocytic sites generally correspond to spectrin-free regions. The methodological quality and imaging data are excellent and represent a strength of the study.
Major comments
The manuscript presents compelling imaging, but several major claims require additional experimental evidence or clarification. The most critical issue concerns the distinction between synaptic and non-synaptic exocytic events. In Figure 1, synaptophysin is used to define synapses, but this is insufficient since synaptophysin is a presynaptic marker and does not confirm the presence of a postsynaptic compartment. The classification of exocytic events as synaptic, therefore, requires co-localization with postsynaptic markers such as PSD95 or Homer. Without this, the paper's main conceptual distinction is not fully supported. Figure 6 requires revision because endocytosis needs to be assessed using a synaptic vesicle-specific assay. A synaptotagmin luminal-domain antibody uptake experiment is recommended, as it would allow precise identification of bona fide SV recycling. This is essential to conclude whether the reported endocytic events reflect synaptic vesicle turnover. The nature of the vesicles undergoing exocytosis along the shaft and at the AIS also remains unresolved. It will be essential to determine whether the authors are observing exocytosis of synaptic vesicles (e.g., VGLUT-positive) or large dense-core vesicles (e.g., BDNF-containing). This can be addressed straightforwardly using available pHluorin-tagged constructs (VGLUT-pHluorin, BDNF-pHluorin). Calcium dependence of the AIS exocytic events should be evaluated. Experiments removing extracellular calcium, blocking voltage-gated calcium channels, or depolarizing neurons (for example, with elevated KCl) would clarify whether these correspond to classical calcium-triggered SV fusion. These requested experiments are realistic in scope and can generally be completed in a few weeks. The imaging, analysis, and methodological descriptions are of high quality, although more information on replication, sample size, and statistical treatment would improve reproducibility.
Minor comments
Clarification of the criteria used to classify spectrin gaps versus clathrin clearings would be helpful. Some figure legends require more detailed acquisition parameters. A clearer description of the image registration and alignment steps in the correlative pipeline would improve transparency. Prior literature on non-synaptic axonal exocytosis and on AIS trafficking could be cited more extensively. The figures are generally high quality, and a schematic summarizing the main findings might help readers.
Referees cross-commenting
Our reviews are pretty consistent overall, I think. Major requests relate to calcium/depolarization dependency, and I would like to insist on the synaptic vs extra-synaptic and SV vs LDCV issues.
Significance
General assessment: This is a technically strong study addressing an interesting and timely question in neuronal cell biology. The imaging quality and methodological innovation are clear strengths. Significant limitations include insufficient distinction between synaptic and non-synaptic release, lack of characterization of vesicle identity, and unclear calcium dependence of AIS exocytosis. Addressing these points will significantly improve the rigor and impact of the study. Advance: The work provides new insights into the spatial organization of axonal exocytosis and its relationship to the actin-spectrin skeleton. The correlative imaging pipeline is valuable. However, the conceptual advance depends on resolving the synaptic/non-synaptic distinction and identifying the vesicle populations involved. Audience: The study will primarily interest a specialized audience in cellular neuroscience, membrane trafficking, and axonal biology. With the recommended revisions, it will also appeal to a broader neurobiology readership interested in nanoscale cytoskeletal organization, synaptic physiology, and axonal signaling.
Field of expertise: neuronal membrane trafficking, synaptic vesicle cycling, autophagy and endolysosomal pathways, cytoskeletal organization. I do not claim expertise in advanced optical engineering, but feel comfortable evaluating the biological interpretations and trafficking mechanisms.
-
