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  1. Evaluation Summary:

    Catsburg et al. provide a new descriptive characterization of clathrin structures in spines vs dendrites using an excellent knock-in approach they recently developed. These results carefully validate earlier findings using the CRISPR approach and constitute useful baseline information, which would be useful in examining changes in the zone induced by neuronal activity or synaptic plasticity.

    (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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  2. Reviewer #3 (Public Review):

    In this MS, the authors first reproduce with some additional details, in particular using various superresolution approaches, our knowledge of the peri-synaptic localization of clathrin labelled endocytic zones (EZ) and determine that peri-synaptic EZ are dynamically distinct from shaft clathrin-coated structures, being more stable. They then perform an extensive characterization of the localization of a set of endocytic accessory proteins and show that a large fraction localizes to the perisynapse. These include HIP1R, ꞵ2-adaptin, Dyn2, CPG2, Eps15, and Itsn1L. In contrast, a subset of other endocytic proteins (PICALM, Epsn2, Amph and FCHO1) were less enriched at EZ. With time lapse video-microscopy, they analyze the residence of these various endocytic proteins next to the PSD and find that they exhibit a range of behavior, from short lived proteins such as FCHO1 to proteins more stably associated to the PSD.

    Next, using two-color single molecule localization microscopy, the authors study the colocalization of these various proteins with respect to clathrin in EZ, and find that endocytic proteins have distinct spatial organization relative to the clathrin structure marking the EZ. They find that ꞵ2-adaptin, Eps15, and Itsn1L were often distributed in smaller patches around and sometimes within the EZ. HIP1R showed a more homogenous distribution and often colocalized with the EZ. Dyn2 showed an overall more homogenous distribution, similar to HIP1R.

    Finally, they touch upon the mechanism of EZ localization next to the PSD. They first recapitulate the central role of Shank by finding that Shank-KD also reduced the association of the PSD with other endocytic proteins in addition to clathrin. They then use a pharmacological approach to suggest that EZ positioning is not related to actin dynamics, and overexpressed an AP2 mutant that unable to interact with PIP2 to suggest that interaction with the membrane is not necessary for EZ positioning.

    Altogether this is a carefully performed study, but the knowledge acquired remains relatively incremental to our previous understanding of EZ positioning.

    The identification of proteins of the endocytic machinery localized at EZ in not surprising and lacks mechanical insight. The study of the mechanism of EZ positioning next to the PSD also lacks additional insight compared to our previous knowledge of the implication of Shank. Finally, this study does not address either the mechanism of EZ assembly nor its function, nor its regulation, which are to my sense the important questions to answer regarding EZs.

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  3. Reviewer #2 (Public Review):

    Catsburg et al attack an interesting topic with a combination of advanced molecular and imaging approaches. The endocytic zone in neuronal spines congregates the endocytic protein clathrin near synapses, and has been demonstrated in several studies to be of functional importance for regulating synaptic transmission by influencing the endocytosis and recycling of glutamate receptors. Basic characterization of this "zone" has been provided by these authors and others, but a more complete description of its character or components has been essentially missing in the field.

    The work first provides new descriptive characterization of clathrin structures in spines vs dendrites using an excellent knock-in approach they recently developed. These results are straightforward but important validation of earlier findings here using the CRISPR approach, and also will be useful baseline information with which to examine changes in the zone for instance during various forms of neuronal plasticity. Then, taking what seems like a tip from the work in other cells as to the organization of proteins at sites of clathrin-mediated endocytosis, they survey the abundance and stability vs transience of a large set of relevant proteins. This is fundamental information necessary to discern the nature and role of the zone.

    The authors then test two mechanisms that might hold the zone or its proteins in place, connections to the PSD protein Shank or to the abundant spine actin cytoskeleton. Experiments to manipulate these two connections do not demonstrate unraveling of the entire system, but instead show a fairly remarkable and specific loss of different proteins. Most notably (to this reader), after Shank knockdown, beta2 adaptin (part of the AP2 complex that was originally used to define the zone in EM) is lost, but Dyn2 (a cytosolic and dynamically recruited part of the machinery during CME) is retained. These and the related results provide new insight to the complex mechanisms that must be at work governing the assembly and dynamics of this specialized domain in spines.

    Overall, the work is systematically and rigorously conducted, and the results are nicely presented with a minor few exceptions. The knock-in application will help establish this general approach as the new standard in the field. Indeed, the overexpression approach used for the protein survey by contrast with the elegant knock-ins feels slightly disappointing, indicating how important it is that the field move as quickly as possible to utilize knock-ins where possible. The overall conclusions about the components, dynamics, and mechanisms of the endocytic zone are stated carefully, and add greatly to understanding of this structure.

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  4. Reviewer #1 (Public Review):

    The authors characterize the molecular organization of the endocytic zone (EZ) known to associate PSD with clathrin coats using advanced imaging approaches, including high-resolution STED imaging and time-lapse live-cell imaging. They also test 12 well-known endocytic proteins to see whether and how they colocalize with EZ and find that a subset of them show strong and long-lived colocalizations, whereas others show weak and transient colocalizations, with EZ by advanced imaging and cell biological disruptions. They find Shank is a key component of the postsynaptic density that coordinates the targeting and localization of the new EZ components. This is a careful and comprehensive analysis of the molecular composition and dynamics of known and additional EZ components using high-resolution and time-lapse imaging. The results are largely convincing and conclusive and provide insights into how synaptic membrane proteins and receptors are trafficked in and out of synapses to regulated synaptic plasticity, which would have significant impacts on broad fields of molecular and cellular neuroscience.

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