DELE1 promotes translation-associated homeostasis, growth, and survival in mitochondrial myopathy.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1's protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody-coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT-EVs. Immunoblot analysis demonstrated KIT-EVs have a distinct protein profile from KIT-depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT-EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT-EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle- and exosome-like EVs. Further, selective KIT-immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.

Unique Aggregation of Retroviral Particles Pseudotyped with the Delta Variant SARS-CoV-2 Spike Protein.

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped viral particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped viral particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike's unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.

The Delta variant SARS-CoV-2 spike protein uniquely promotes aggregation of pseudotyped viral particles.

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike’s unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

CD47 interactions with exportin-1 limit the targeting of m7G-modified RNAs to extracellular vesicles.

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5'-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex.

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

The endosomal recycling system dynamically tunes synaptic strength, which underlies synaptic plasticity. Exocytosis is involved in the expression of long-term potentiation (LTP), as postsynaptic cleavage of the SNARE (soluble NSF-attachment protein receptor) protein VAMP2 by tetanus toxin blocks LTP. Moreover, induction of LTP increases the exocytosis of transferrin receptors (TfRs) and markers of recycling endosomes (REs), as well as post-synaptic AMPA type receptors (AMPARs). However, the interplay between AMPAR and TfR exocytosis remains unclear. Here, we identify VAMP4 as the vesicular SNARE that mediates most dendritic RE exocytosis. In contrast, VAMP2 plays a minor role in RE exocytosis. LTP induction increases the exocytosis of both VAMP2- and VAMP4-labeled organelles. Knock down (KD) of VAMP4 decreases TfR recycling but increases AMPAR recycling. Moreover, VAMP4 KD increases AMPAR-mediated synaptic transmission, which consequently occludes LTP expression. The opposing changes in AMPAR and TfR recycling upon VAMP4 KD reveal their sorting into separate endosomal populations.

A Bimodal Nanosensor for Probing Influenza Fusion Protein Activity Using Magnetic Relaxation.

Viral fusion is a critical step in the entry pathway of enveloped viruses and remains a viable target for antiviral exploration. The current approaches for studying fusion mechanisms include ensemble fusion assays, high-resolution cryo-TEM, and single-molecule fluorescence-based methods. While these methods have provided invaluable insights into the dynamic events underlying fusion processes, they come with their own limitations. These often include extensive data and image analysis in addition to experimental time and technical requirements. This work proposes the use of the spin-spin T2 relaxation technique as a sensitive bioanalytical method for the rapid quantification of interactions between viral fusion proteins and lipids in real time. In this study, new liposome-coated iron oxide nanosensors (LIONs), which mimic as magnetic-labeled host membranes, are reported to detect minute interactions occurring between the membrane and influenza's fusion glycoprotein, hemagglutinin (HA). The influenza fusion protein's interaction with the LION membrane is detected by measuring changes in the sensitive spin-spin T2 magnetic relaxation time using a bench-top NMR instrument. More data is gleaned from including the fluorescent dye DiI into the LION membrane. In addition, the effects of environmental factors on protein-lipid interaction that affect fusion such as pH, time of incubation, trypsin, and cholesterol were also examined. Furthermore, the efficacy and sensitivity of the spin-spin T2 relaxation assay in quantifying similar protein/lipid interactions with more native configurations of HA were demonstrated using virus-like particles (VLPs). Shorter domains derived from HA were used to start a reductionist path to identify the parts of HA responsible for the NMR changes observed. Finally, the known fusion inhibitor Arbidol was employed in our spin-spin T2 relaxation-based fusion assay to demonstrate the application of LIONs in real-time monitoring of this aspect of fusion for evaluation of potential fusion inhibitors.

Structural basis for placental malaria mediated by Plasmodium falciparum VAR2CSA.

Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment. In the present study, we determined the cryo-electron microscopy structures of the full-length ectodomain of VAR2CSA from P. falciparum strain NF54 in complex with CSA, and VAR2CSA from a second P. falciparum strain FCR3. The architecture of VAR2CSA is composed of a stable core flanked by a flexible arm. CSA traverses the core domain by binding within two channels and CSA binding does not induce major conformational changes in VAR2CSA. The CSA-binding elements are conserved across VAR2CSA variants and are flanked by polymorphic segments, suggesting immune selection outside the CSA-binding sites. This work provides paths for developing interventions against placental malaria and cancer.

Identifying SARS-CoV-2 Entry Inhibitors through Drug Repurposing Screens of SARS-S and MERS-S Pseudotyped Particles.

While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here, inhibitors of spike (S) mediated cell entry were identified in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. We discovered six compounds (cepharanthine, abemaciclib, osimertinib, trimipramine, colforsin, and ingenol) to be broad spectrum inhibitors for spike-mediated entry. This work could contribute to the development of effective treatments against the initial stage of viral infection and provide mechanistic information that might aid the design of new drug combinations for clinical trials for COVID-19 patients.

Macrophage-derived HIV-1 carries bioactive TGF-beta.

Infected T cells and macrophages are the main producers of HIV-1 in infected individuals. Upon release from infected cells, HIV-1 incorporates various cellular membrane proteins, some of which are specific for these cells. However, the functions of cell-encoded proteins in virions remain largely unknown. We performed flow virometry to identify, in plasma of HIV-infected individuals, macrophage- and T-cell-derived HIV-1 virions, using cell-specific markers CD36 and CD27, respectively. Using four different methods, we demonstrated that CD36 on virions binds the immunosuppressive cytokine transforming growth factor beta (TGF-ß) through a ligand, thrombospondin one (TSP-1). Flow virometry of individual virions showed that TGF-ß was present on CD36+ virions (average, 28.2% ± 6.6% (n = 3)) but not on CD27+ virions (average, 1% ± 0.1% (n = 3)). TGF-ß molecules present on captured CD36+ virions were biologically active, as evaluated with a reporter cell line. Delivery of TGF-ß on HIV virions to HIV target cells may affect them, playing a significant role in viral pathogenesis.

Neuronal Activity and Intracellular Calcium Levels Regulate Intracellular Transport of Newly Synthesized AMPAR.

Regulation of AMPA receptor (AMPAR) trafficking is a key modulator of excitatory synaptic transmission; however, intracellular vesicular transport of newly synthesized AMPARs has been little studied due to technical limitations. By combining molecular tools with imaging strategies in cultured rat hippocampal neurons, we found that vesicles containing newly synthesized, GluA1-subunit-containing AMPARs are transported antero- and retrogradely at a mean speed of 1.5 µm.s-1. Synaptic activity and variations in intracellular calcium levels bidirectionally modulate GluA1 transport. Chemical long-term potentiation (cLTP) initially induces a halt in GluA1 transport, followed by a sustained increase, while acute glutamate uncaging on synaptic spines arrests vesicular movements. GluA1 phosphomimetic mutants preferentially travel to the dendritic tip, probably to replenish extrasynaptic pools, distal to the soma. Our findings indicate that AMPAR intracellular transport is highly regulated during synaptic plasticity and likely controls AMPAR numbers at the plasma membrane.

Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons.

Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons. We found that zinc is distributed all along the dendrites while copper is mainly pinpointed within the spines. In spines, zinc content is higher within the spine head while copper is higher within the spine neck. Such specific distributions suggested metal interactions with cytoskeleton proteins. Zinc supplementation induced the increase of ß-tubulin content in dendrites. Copper supplementation impaired the ß-tubulin and F-actin networks. Copper chelation resulted in the decrease of F-actin content in dendrites, drastically reducing the number of F-actin protrusions. These results indicate that zinc is involved in microtubule stability whereas copper is essential for actin-dependent stability of dendritic spines, although copper excess can impair the dendritic cytoskeleton.

Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity.

Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission.

A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks.

Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines.

Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process.

Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95.

The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resolution light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concentrated inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them. Nanodomains are dynamic in their shape and position within synapses and can form or disappear within minutes, although they are mostly stable for up to 1 h. AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95, which are generally of larger size than AMPAR nanoclusters. PSD95 expression level regulates AMPAR nanodomain size and compactness in parallel to miniature EPSC amplitude. Monte Carlo simulations further indicate the impact of AMPAR concentration in clusters on the efficacy of synaptic transmission. The observation that AMPARs are highly concentrated in nanodomains, instead of diffusively distributed in the PSD as generally thought, has important consequences on our understanding of excitatory neurotransmission. Furthermore, our results indicate that glutamatergic synaptic transmission is controlled by the nanometer-scale regulation of the size of these highly concentrated nanodomains.

Mass of the postsynaptic density and enumeration of three key molecules.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.