Dynamics and nanoscale organization of the postsynaptic endocytic zone at excitatory synapses

Abstract

At postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy to reveal the dynamic organization of this poorly understood clathrin structure in rat hippocampal neurons. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting interactions with the actin cytoskeleton or membrane did not alter EZ positioning. Finally, we found that plasticity-inducing stimuli promoted the reorganization of the EZ. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.

Living on the edge

“…They observed that proteins contributing to the early phase of endocytosis mostly accumulate specifi cally at the periphery of the EZ. While other endocytic proteins, mostly involved in the later phase of endocytosis including vesicle scission, were more widely and centrally distributed at the EZ (Catsburg et al, 2022), consistent with observations at clathrin sheets in non-neuronal cells (Sochacki et al, 2017). Also, while some endocytic proteins such as CPG2 were found to contribute more generally to receptor internalization, others such as Endophilin-B2 were found specifi cally required for activity-induced, but not constitutive internalization (Loebrich et al, 2016).…”

“…Also, while some endocytic proteins such as CPG2 were found to contribute more generally to receptor internalization, others such as Endophilin-B2 were found specifi cally required for activity-induced, but not constitutive internalization (Loebrich et al, 2016). Remarkably, the EZ undergoes activity-induced remodeling to accommodate long-term changes in synaptic strength (Catsburg et al, 2022). This suggests that reorganization of the EZ induced upon distinct synaptic activity patterns facilitates changes in endocytic rate of receptors modulating long-term synaptic plasticity, a hypothesis that would be interesting to test.…”

“…For mGluR5 this may be regulated via β-arrestin since activated mGluR5 is desensitized by the recruitment of β-arrestin that targets the 7 receptor for internalization via clathrin-mediated endocytosis, but also acts as an adaptor for the endocytic machinery components AP-2 and Clathrin (Ferguson et al, 1996;. AP-2, like Clathrin, localizes to the EZ and is known to bind to cytosolic receptor domains and might partake in the perisynaptic positioning of mGluRs (Catsburg et al, 2022;. Many questions remain however; do mGluR5 nanodomains colocalize with the perisynaptic EZ?…”

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells

“…Strep-KDEL-Halo-Transferrin Receptor (TfR)-SBP (Halo-RUSH-TfR) (called RUSH-TfR in the text) was provided by Dr. Jennifer Lippincott-Schwartz (Weigel et al, 2021) (Addgene plasmid #166905) pF282-hEF1a-H2B-mNeonGreen-IRES-Puro x Tol2 was a gift from Dr. Judith Klumperman. Halo-Clathrin (Catsburg et al, 2022) was a gift from Dr. Harold MacGillavry.…”

Sodium channel endocytosis drives axon initial segment plasticity

“…The knock-in constructs of GluN1-GFP and clathrin light chain A-GFP (Willems et al, 2020), and dynamin2-GFP (Catsburg, Westra, van Schaik, & MacGillavry, 2022) were previously described. The knock-in construct for Nav1.2 was cloned in the pORANGE (Willems et al, 2020) and the gRNA used was: GGACAAAGGGAAAGATATCA.…”