IRSp53 accumulates at the postsynaptic density under excitatory conditions

Döṡemeci, Ayṡe; Burch, Amelia; Loo, Hannah; Toy, Dana; Tao-Cheng, Jung-Hwa

doi:10.1371/journal.pone.0190250

Cited by 13 publications

(13 citation statements)

References 27 publications

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“…These proteins include CaMKII, Shank, IRSp53, and CYLD. The most likely major contributor for the increased staining of this layer is CaMKII because of its great abundance at the PSD [12, 13] and its substantial increase (~ 3 fold) under excitatory conditions [6, 7], whereas Shank and IRSp53 increase only ~ 1.5 fold over controls [14–16]. CYLD, although greatly increased at the PSD upon stimulation [17], has an overall low abundance compared to these other PSD proteins [13].…”

Section: Discussionmentioning

confidence: 99%

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures

Tao-Cheng

2019

Mol Brain

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Activity can induce structural changes in glutamatergic excitatory synapses, including increase in thickness and curvature of the postsynaptic density (PSD); these structural changes can only be documented by electron microscopy. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, increases in thickness and curvature of PSDs were noticeable within 30 s of stimulation and progressed with time up to 3 min. These structural changes were reversible upon returning the samples to control medium for 5–10 min. Thus, the postsynaptic density is a very dynamic structure that undergoes rapid reorganization of its components upon stimulation, and recovery upon cessation of stimulation. The gradual increase in thickness of PSD could result from a gradual translocation of some PSD proteins to the PSD, and the increase in curvature of the PSD is likely led by postsynaptic elements. Electronic supplementary material The online version of this article (10.1186/s13041-019-0468-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures

Tao-Cheng

2019

Mol Brain

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“…Clusters of synaptic vesicles (SV in a ) and the postsynaptic density (PSD, edges of which marked with arrows in a ) are characteristics of glutamatergic asymmetric synapses. The fixation treatments were 4% PF in PBS for 45 min ( a ), 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 30 min ( b ), 4% PF in PBS for 30 min and labeled for CaMKII ( c ), a cytosolic protein in neurons [ 6 ], and 2% PF in PBS for 15 min and labeled for IRSp53 ( d ), an actin-associated protein enriched at the PSD [ 20 ]. All fixations were carried out at room temperature.…”

Section: Resultsmentioning

confidence: 99%

“…2 c. However, even though membranes were not optimally preserved in this lesser-fixed sample (Fig. 2 d), the resulting labeling at the postsynaptic compartment is still credible and quantification of labeling density is valid [ 20 ].…”

Section: Resultsmentioning

confidence: 99%

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

et al. 2021

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Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

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“…However, the amount of CYLD at the PSD is only~8% of that of Shank 3 [27], making CYLD a minor player in CaMKII binding at the PSD compared to Shank 3. On the other hand, there is no evidence of Homer and IRSp53, two other PSD proteins that are located in the PSD pallium [28,29], interacting directly with CaMKII.…”

Section: Discussionmentioning

confidence: 97%

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons

Tao-Cheng

2020

Mol Brain

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Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K + or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within~30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core,~100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

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IRSp53 accumulates at the postsynaptic density under excitatory conditions

Cited by 13 publications

References 27 publications

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons

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