Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking
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Evaluation Summary:
In this manuscript, the authors developed a sensitive single particle tracking method for endogenous AMPA receptors. They found that AMPAR-containing vesicles showed reduced mobility near stimulation sites, likely due to increased F-actin bundling in dendritic shafts. The study found a novel mechanism of AMPAR trafficking using state-of-the-art labeling and analysis techniques, and thus will be of great interest for broad audience. However, their conclusion requires additional experimental support.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.
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Author response:
Reviewer #1 (Public Review):
Authors propose a mechanism where actin polymerization in the dendritic shaft plays a key role in trapping AMPAR vesicles around the stimulated site, promoting the preferential insertion of AMPAR into the potentiated synapse. This dendritic mechanism is novel and may be important for phenomena. Authors also developed a sophisticated method to observe the endogenous behavior of AMPAR using the HITI system.
However, there are some major issues that need to be addressed to support the authors' claims. Also, overall, it is hard to follow. It could be better written.
We thank the reviewer for carefully reading our text and for the helpful recommendations. We have performed additional experiments and analysis to address the raised issues (detailed below). In addition, we have streamlined and …
Author response:
Reviewer #1 (Public Review):
Authors propose a mechanism where actin polymerization in the dendritic shaft plays a key role in trapping AMPAR vesicles around the stimulated site, promoting the preferential insertion of AMPAR into the potentiated synapse. This dendritic mechanism is novel and may be important for phenomena. Authors also developed a sophisticated method to observe the endogenous behavior of AMPAR using the HITI system.
However, there are some major issues that need to be addressed to support the authors' claims. Also, overall, it is hard to follow. It could be better written.
We thank the reviewer for carefully reading our text and for the helpful recommendations. We have performed additional experiments and analysis to address the raised issues (detailed below). In addition, we have streamlined and shortened the text to improve its clarity and focus on the biological story.
Reviewer #2 (Public Review):
In this study, Wong and colleagues investigate mechanisms leading to input-specificity of LTP. They focus on the trafficking of AMPA receptors as the surface accumulation of AMPARs is one of the key features of potentiated synapses. They employ an elegant strategy to label endogenous GluA1 with a HaloTag using CRISPR-based technology and succeed to find targeting site which does not interfere with receptor's trafficking or function. This allowed them to visualize and track single receptors in endosomes as well as at the plasma membrane of primary rat hippocampal neurons. They develop and extend particle tracking and molecule counting algorithms to analyze active transport and diffusion of AMPARs and, as expected find that neuronal activation leads to increased surface expression of labelled AMPARs. Interestingly, they also observe a strong decrease in long-range motion of AMPAR-containing vesicles upon induction of chemical LTP. From this point, the manuscript focuses on explaining this observation. The authors switch from a global activation protocol to glutamate uncaging to induce LTP at individual synapses. Also, in these settings, they measure the reduction in mobile vesicle fraction within about 30 µm long dendritic segment containing the activated spine. In search of an explanation, they investigate activity-dependent actin polymerization as a possible confinement factor that could change the motility of organelles in dendrites. Their hypotheses is based on pre-existing literature demonstrating the role of F-actin in trapping and stalling dendritic endolysosomes as well similar role of F-actin in non-neuronal cells. Indeed, the authors convincingly show that pharmacological depolymerization or stabilization of F-actin bidirectionally impacts the trafficking behavior of AMPAR-containing vesicles in the dendritic shaft. To directly visualize effects of structural LTP at individual synapses on dendritic actin cytoskeleton, they employ a F-actin-binding probe Tractin. Here they find that cLTP results in the formation of dendritic F-actin fibers and bundles arranged in a network. The spatial extent of such a network correlates with an area where AMPAR vesicles exhibit decreased motility. Although this makes sense, I have some concerns about these experiments.
Tractin has been previously published as F-actin marker but like several other binding probes (i.e. lifeact), it affects F-actin structure and dynamics. The large number of F-actin bundles is not very typical for dendrites of hippocampal neurons and might be an artifact of Tractin overexpression. It is difficult to judge whether this is a case because there is no comparison with the endogenous situation where F-actin is labelled directly. The final series of experiments focus on the role of processive myosins in stalling and exocytosis of AMPAR vesicles. To address this point, the authors employ a mixture of three different myosin inhibitors and show that although myosins are not responsible for increased vesicle confinement they facilitate exocytosis of AMPARs. What I find somewhat missing are data and examples of AMPAR trafficking into dendritic spines. Also here, stronger experimental support could benefit the conclusions.
Overall, the authors achieved the aims of their study. They demonstrated that synapse-specific potentiation results in signaling which triggers actin polymerization in dendritic shaft beneath the activated input. This leads to trapping and accumulation of AMPAR-containing endosomes which then have higher probability to be delivered and secreted at activated dendritic spines. In addition to conceptual advance of this work, several state-of-the-art labeling and analysis techniques where developed in this project and they will likely be used by other groups.
We thank the reviewer for raising these important issues with regards to the use of tractin as a marker for actin polymerization. We have performed additional experiments (detailed below) using phalloidin and also dominant negative inhibitors of myosin Va, Vb, and VI in order to strengthen our conclusions. We find that inducing synaptic activity with cLTP increases phalloidin labeling and the appearance of F-actin fibers. Moreover, inhibition of myosin Va and Vb (but not VI) using their dominant negative c-terminal domains recapitulates the effects of pharmacological inhibition on both the motion states and directional bias of GluA1-HT vesicles in response to cLTP.
With regards to AMPAR trafficking into spines, we and others have found that GluA1-containing vesicles rarely enter dendritic spines (see response to Reviewer #2, comment 3). Furthermore, exocytic events occur largely at extrasynaptic sites, such as on the dendritic shaft (Figure 5-video 1-3; Lin et al., 2007; Makino et al., 2009; Patterson et al., 2010). Consequently, we believe vesicles are concentrated proximal to synaptic activity in the dendritic shaft rather than in the dendritic spine itself, creating a larger reservoir of intracellular AMPARs that can exocytose during synaptic activity. Others have demonstrated that surface bound AMPARs diffuse across the cell membrane into stimulated synapses where they are captured (Choquet and Opazo, 2022).
We also thank the reviewers for acknowledging the conceptual and technical advances in this work.
Reviewer #3 (Public Review):
Wong et al. developed a new versatile approach with a robust signal to track protein dynamics by inserting a tag into the endogenous loci and different properties of fluorescent dyes for conjugation. Using this approach, the authors monitor the trafficking of Fluorescent dye and Halo-tagged GluA1 with time-lapse imaging and found that neuronal stimulation induces GluA1 accumulation surrounding stimulated synapses on dendritic shafts and actin polymerization at synapses and dendrites. Furthermore, combining with pharmacological manipulations of actin polymerization or myosin activity, the authors found that actin polymerization facilitates exocytosis of GluA1 near activated synapses. The new approach may provide broad impacts upon appropriate control experiments, and the practical application of this approach to GluA1 trafficking upon neuronal activation is significant. However, there are several weaknesses, including confirmation of activity of the tagged receptors and receptor specificity mimicking endogenous LTP machinery. If the receptor tagged by the new robust approach reflects endogenous machinery, this approach will provide a big opportunity to the community as a versatile method to visualize a protein not visualized previously.
Although we use methods previously demonstrated to stimulate LTP, we do not ourselves demonstrate LTP using electrophysiological methods, and consequently we have changed the text to focus on synaptic plasticity (specifically structural plasticity). Furthermore, we confirm the activity of HaloTag knock-in receptors by expressing GluA1-HT and GluA1-HT-SEP in HEK293T cells and performing whole-cell patch clamp experiments. We find that GluA1-HT and GluA1-HT-SEP responds to glutamate in a similar manner to untagged GluA1.
We also thank the reviewer for acknowledging the novelty of our strategy.
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Evaluation Summary:
In this manuscript, the authors developed a sensitive single particle tracking method for endogenous AMPA receptors. They found that AMPAR-containing vesicles showed reduced mobility near stimulation sites, likely due to increased F-actin bundling in dendritic shafts. The study found a novel mechanism of AMPAR trafficking using state-of-the-art labeling and analysis techniques, and thus will be of great interest for broad audience. However, their conclusion requires additional experimental support.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
Authors propose a mechanism where actin polymerization in the dendritic shaft plays a key role in trapping AMPAR vesicles around the stimulated site, promoting the preferential insertion of AMPAR into the potentiated synapse. This dendritic mechanism is novel and may be important for phenomena. Authors also developed a sophisticated method to observe the endogenous behavior of AMPAR using the HITI system.
However, there are some major issues that need to be addressed to support the authors' claims. Also, overall, it is hard to follow. It could be better written.
-
Reviewer #2 (Public Review):
In this study, Wong and colleagues investigate mechanisms leading to input-specificity of LTP. They focus on the trafficking of AMPA receptors as the surface accumulation of AMPARs is one of the key features of potentiated synapses. They employ an elegant strategy to label endogenous GluA1 with a HaloTag using CRISPR-based technology and succeed to find targeting site which does not interfere with receptor's trafficking or function. This allowed them to visualize and track single receptors in endosomes as well as at the plasma membrane of primary rat hippocampal neurons. They develop and extend particle tracking and molecule counting algorithms to analyze active transport and diffusion of AMPARs and, as expected find that neuronal activation leads to increased surface expression of labelled AMPARs. …
Reviewer #2 (Public Review):
In this study, Wong and colleagues investigate mechanisms leading to input-specificity of LTP. They focus on the trafficking of AMPA receptors as the surface accumulation of AMPARs is one of the key features of potentiated synapses. They employ an elegant strategy to label endogenous GluA1 with a HaloTag using CRISPR-based technology and succeed to find targeting site which does not interfere with receptor's trafficking or function. This allowed them to visualize and track single receptors in endosomes as well as at the plasma membrane of primary rat hippocampal neurons. They develop and extend particle tracking and molecule counting algorithms to analyze active transport and diffusion of AMPARs and, as expected find that neuronal activation leads to increased surface expression of labelled AMPARs. Interestingly, they also observe a strong decrease in long-range motion of AMPAR-containing vesicles upon induction of chemical LTP. From this point, the manuscript focuses on explaining this observation. The authors switch from a global activation protocol to glutamate uncaging to induce LTP at individual synapses. Also, in these settings, they measure the reduction in mobile vesicle fraction within about 30 µm long dendritic segment containing the activated spine. In search of an explanation, they investigate activity-dependent actin polymerization as a possible confinement factor that could change the motility of organelles in dendrites. Their hypotheses is based on pre-existing literature demonstrating the role of F-actin in trapping and stalling dendritic endolysosomes as well similar role of F-actin in non-neuronal cells. Indeed, the authors convincingly show that pharmacological depolymerization or stabilization of F-actin bidirectionally impacts the trafficking behavior of AMPAR-containing vesicles in the dendritic shaft. To directly visualize effects of structural LTP at individual synapses on dendritic actin cytoskeleton, they employ a F-actin-binding probe Tractin. Here they find that cLTP results in the formation of dendritic F-actin fibers and bundles arranged in a network. The spatial extent of such a network correlates with an area where AMPAR vesicles exhibit decreased motility. Although this makes sense, I have some concerns about these experiments.
Tractin has been previously published as F-actin marker but like several other binding probes (i.e. lifeact), it affects F-actin structure and dynamics. The large number of F-actin bundles is not very typical for dendrites of hippocampal neurons and might be an artifact of Tractin overexpression. It is difficult to judge whether this is a case because there is no comparison with the endogenous situation where F-actin is labelled directly. The final series of experiments focus on the role of processive myosins in stalling and exocytosis of AMPAR vesicles. To address this point, the authors employ a mixture of three different myosin inhibitors and show that although myosins are not responsible for increased vesicle confinement they facilitate exocytosis of AMPARs. What I find somewhat missing are data and examples of AMPAR trafficking into dendritic spines. Also here, stronger experimental support could benefit the conclusions.
Overall, the authors achieved the aims of their study. They demonstrated that synapse-specific potentiation results in signaling which triggers actin polymerization in dendritic shaft beneath the activated input. This leads to trapping and accumulation of AMPAR-containing endosomes which then have higher probability to be delivered and secreted at activated dendritic spines. In addition to conceptual advance of this work, several state-of-the-art labeling and analysis techniques where developed in this project and they will likely be used by other groups.
-
Reviewer #3 (Public Review):
Wong et al. developed a new versatile approach with a robust signal to track protein dynamics by inserting a tag into the endogenous loci and different properties of fluorescent dyes for conjugation. Using this approach, the authors monitor the trafficking of Fluorescent dye and Halo-tagged GluA1 with time-lapse imaging and found that neuronal stimulation induces GluA1 accumulation surrounding stimulated synapses on dendritic shafts and actin polymerization at synapses and dendrites. Furthermore, combining with pharmacological manipulations of actin polymerization or myosin activity, the authors found that actin polymerization facilitates exocytosis of GluA1 near activated synapses. The new approach may provide broad impacts upon appropriate control experiments, and the practical application of this approach …
Reviewer #3 (Public Review):
Wong et al. developed a new versatile approach with a robust signal to track protein dynamics by inserting a tag into the endogenous loci and different properties of fluorescent dyes for conjugation. Using this approach, the authors monitor the trafficking of Fluorescent dye and Halo-tagged GluA1 with time-lapse imaging and found that neuronal stimulation induces GluA1 accumulation surrounding stimulated synapses on dendritic shafts and actin polymerization at synapses and dendrites. Furthermore, combining with pharmacological manipulations of actin polymerization or myosin activity, the authors found that actin polymerization facilitates exocytosis of GluA1 near activated synapses. The new approach may provide broad impacts upon appropriate control experiments, and the practical application of this approach to GluA1 trafficking upon neuronal activation is significant. However, there are several weaknesses, including confirmation of activity of the tagged receptors and receptor specificity mimicking endogenous LTP machinery. If the receptor tagged by the new robust approach reflects endogenous machinery, this approach will provide a big opportunity to the community as a versatile method to visualize a protein not visualized previously.
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