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At least 1,423 records · Page 79
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Quantifying the effect of multiple load flexibility strategies on commercial building electricity demand and services via surrogate modeling

The expansion of commercial building demand response as a demand-side management resource for the electric grid necessitates new decision support resources for customers seeking to assess the benefit–risk tradeoffs of possible strategies for energy flexible building operations. To address this need, we, in this study, develop surrogate models that predict the impacts of several load flexibility strategies on commercial building electricity demand and indoor temperature, focusing on offices and retail buildings at multiple scales. The surrogate models are fit to a synthetic database generated via whole building simulations, which establish the relationships between the key operational features of a given strategy and potential changes in building demand and temperature across a variety of contexts. The surrogate models are translated to a Bayesian framework to allow straightforward communication of uncertainty and parameter updating given new evidence. We find strong predictive performance across the suite of models, underscoring the usefulness of the approach in guiding decisions about implementing load flexibility strategies under a particular set of operational and environmental conditions.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2022

Evaluation of artificial neural network performance for classification of potato plants infected with potato virus Y using spectral data on multiple varieties and genotypes

Potato virus Y (Potyviridae, PVY) is a plant virus that poses a significant threat to potato producers on a global basis. The pathogen has disrupted seed potato supplies and negatively impacted yield and quality of commercial potato crops. The potato industry currently manages PVY infection levels via insecticide applications, regional seed certification programs that rely on field scouting to visually assess individual plants for infection status, and destructive and costly tissue sampling coupled with laboratory assays. Despite these efforts, PVY continues to confound potato industry stakeholders resulting in economic harm. Remote sensing and machine learning provide for the development of new tools to more accurately detect and spatially quantify PVY-infected plants versus the current state of the art. However, there is a need to understand how the occurrence of many different potato varieties impact the dynamics of developing models to detect potato plants impacted with PVY and their potential effectiveness. This study evaluates classification modelling outcomes using spectral datasets collected in different temporal and spatial environments (greenhouse and a production field) on multiple potato varieties consisting of labelled instances of plants infected with PVY and those not infected with the virus. A modelling framework was developed to support iterative modelling runs using artificial neural network (ANN) architectures configured as binary classifiers to develop sample populations to support statistical analysis on model performance using specific spectral subsets. When using spectral data to detect PVY-infected plants, ANN models achieved the highest mean accuracy of 0.894 on a single variety. Conversely, the same ANN model architecture only achieved a mean accuracy of 0.575 on a spectral data set representing 29 potato breeding lines. Additionally, statistical analysis indicates spectral regions including the red edge, near infrared and shortwave infrared contain more important spectral features for the ANN classifier introduced in this research.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Quantitative evaluations of subtropical westerly jet simulations over East Asia based on multiple CMIP5 and CMIP6 GCMs

As a salient feature of the Asian monsoon system, the East Asian subtropical westerly jet (EASWJ) exerts significant impacts on weather and climate changes in China and even throughout East Asia. In this paper, we applied a new self-adaptive algorithm to detect the EASWJ, identify its boundaries, and then represent its characteristics by defining three indices: the intensity index, meridional displacement index, and width index. Compared to the reanalysis data, we carried out a comprehensive, objective, and quantitative EASWJ evaluation using historical experiments from multiple global climate models (GCMs) in the Coupled Model Intercomparison Project Phases 5 and 6 (CMIP5 and CMIP6). The results show that the multimodel ensemble mean (MME) of both CMIP5 and CMIP6 can simulate the characteristics of winter EASWJ well. While for the other three seasons, the MME of both phase models underestimate the 200-hPa zonal wind (U200) strength in the jet coverage area and overestimate the U200 outside the jet area, such simulation results weaken the meridional shear of the wind field. The EASWJ simulations from the CMIP5 GCMs had no consistent intensity or location characteristic tendencies, and most CMIP5 GCMs tended to simulate relatively wide-coverage jets. In contrast, most CMIP6 GCMs are inclined to simulate significantly weaker, wider, and more-northward jets. Compared to the predecessors in CMIP5, about half of CMIP6 GCMs significantly minimized the jet intensity bias, but remarkable errors were still observed in their jet location and coverage representations. Furthermore, the comparative analysis performed by classifying models based on their evaluated simulation results suggested that the simulated performance of the meridional temperature gradient was important for capturing the EASWJ characteristics. Further in-depth study of the causes of model differences is warranted to improve simulation results.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2011

Multiple post-translational modifications in hepatocyte nuclear factor 4{alpha}

Highlights: {yields} We performed comprehensive PTM analysis for HNF4{alpha} protein. {yields} We identified 8 PTMs in HNF4{alpha} protein including newly identified PTMs. {yields} Among them, we found acetylation at lysine 458 was one of the prime PTMs for HNF4{alpha} function. {yields} Acetylation at lysine 458 was inhibitory for HNF4{alpha} transcription function. {yields} This modification fluctuated in response to extracellular condition. -- Abstract: To investigate the role of post-translational modifications (PTMs) in the hepatocyte nuclear factor 4{alpha} (HNF4{alpha})-mediated transcription, we took a comprehensive survey of PTMs in HNF4{alpha} protein by massspectrometry and identified totally 8 PTM sites including newly identified ubiquitilation and acetylation sites. To assess the impact of identified PTMs in HNF4{alpha}-function, we introduced point mutations at the identified PTM sites and, tested transcriptional activity of the HNF4{alpha}. Among the point-mutations, an acetylation site at lysine 458 was found significant in the HNF4{alpha}-mediated transcriptional control. An acetylation negative mutant at lysine 458 showed an increased transcriptional activity by about 2-fold, while an acetylation mimic mutant had a lowered transcriptional activation. Furthermore, this acetylation appeared to be fluctuated in response to extracellular nutrient conditions. Thus, by applying an comprehensive analysis of PTMs, multiple PTMs were newly identified in HNF4{alpha} and unexpected role of an HNF4{alpha} acetylation could be uncovered.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells

Highlights: •A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. •The protein accumulates in the cytosol in an intact form. •The internalized protein is multiply phosphorylated. -- Abstract: Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expression of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

miR-186 inhibits cell proliferation in multiple myeloma by repressing Jagged1

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids that regulate gene expression by targeting mRNAs for translational repression and degradation. Accumulating experimental evidence supports a causal role of miRNAs in hematology tumorigenesis. However, the specific functions of miRNAs in the pathogenesis of multiple myeloma (MM) remain to be established. In this study, we demonstrated that miR-186 is commonly downregulated in MM cell lines and patient MM cells. Ectopic expression of miR-186 significantly inhibited cell growth, both in vitro and in vivo, and induced cell cycle G{sub 0}/G{sub 1} arrest. Furthermore, miR-186 induced downregulation of Jagged1 protein expression by directly targeting its 3′-untranslated region (3′-UTR). Conversely, overexpression of Jagged1 rescued cells from miR-186-induced growth inhibition. Our collective results clearly indicate that miR-186 functions as a tumor suppressor in MM, supporting its potential as a therapeutic target for the disease. - Highlights: • miR-186 expression is decreased in MM. • miR-186 inhibits MM cell proliferation in vitro and in vivo. • Jagged1 is regulated by miR-186. • Overexpression of Jagged1 reverses the effects of miR-186.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3

Aberrant expression of microRNAs (miRNAs) is implicated in cancer development and progression. While miR-320a is reported to be deregulated in many malignancy types, its biological role in multiple myeloma (MM) remains unclear. Here, we observed reduced expression of miR-320a in MM samples and cell lines. Ectopic expression of miR-320a dramatically suppressed cell viability and clonogenicity and induced apoptosis in vitro. Mechanistic investigation led to the identification of Pre-B-cellleukemia transcription factor 3 (PBX3) as a novel and direct downstream target of miR-320a. Interestingly, reintroduction of PBX3 abrogated miR-320a-induced MM cell growth inhibition and apoptosis. In a mouse xenograft model, miR-320a overexpression inhibited tumorigenicity and promoted apoptosis. Our findings collectively indicate that miR-320a inhibits cell proliferation and induces apoptosis in MM cells by directly targeting PBX3, supporting its utility as a novel and potential therapeutic agent for miRNA-based MM therapy. -- Highlights: •Expression of miR-320a in MM cell induces apoptosis in vitro. •miR-320a represses PBX3 via targeting specific sequences in the 3′UTR region. •Exogenous expression of PBX3 reverses the effects of miR-320a in inhibiting MM cell growth and promoting apoptosis. •Overexpression of miR-320a inhibits tumor growth and increases apoptosis in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Epigenetic identification of ZNF545 as a functional tumor suppressor in multiple myeloma via activation of p53 signaling pathway

The KRAB–zinc-finger protein ZNF545 was recently identified as a potential suppressor gene in several tumors. However, the regulatory mechanisms of ZNF545 in tumorigenesis remain unclear. In this study, we investigated the expression and roles of ZNF545 in multiple myeloma (MM). ZNF545 was frequently downregulated in MM tissues compared with non-tumor bone marrow tissues. ZNF545 expression was silenced by promoter methylation in MM cell lines, and could be restored by demethylation treatment. ZNF545 methylation was detected in 28.3% of MM tissues, compared with 4.3% of normal bone marrow tissues. ZNF545 transcriptionally activated the p53 signaling pathway but had no effect on Akt in MM, whereas ectopic expression of ZNF545 in silenced cells suppressed their proliferation and induced apoptosis. We therefore identified ZNF545 as a novel tumor suppressor inhibiting tumor growth through activation of the p53 pathway in MM. Moreover, tumor-specific methylation of ZNF545 may represent an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. -- Highlights: •Downregulated ZNF545 in MM tissues and cell lines and ectopic expression of ZNF545 suppresses tumor growth. •Tumor-specific methylation of ZNF545 represents an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. •ZNF545 exerts its tumor suppressive effects via transcriptional activating p53 pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Nucleocytoplasmic trafficking of Nipah virus W protein involves multiple discrete interactions with the nuclear import and export machinery

Paramyxoviruses replicate in the cytoplasm with no obvious requirement to interact with the nucleus. Nevertheless, the W protein of the highly lethal bat-borne paramyxovirus Nipah virus (NiV) is known to undergo specific targeting to the nucleus, mediated by a single nuclear localisation signal (NLS) within the C-terminal domain. Here, we report for the first time that additional sites modulate nucleocytoplasmic localisation of W. We show that the N-terminal domain interacts with importin α1 and contributes to nuclear accumulation of W, indicative of a novel N-terminal NLS. We also find that W undergoes exportin-1 mediated nuclear export, dependent on a leucine at position 174. Together, these data enable significant revision of the generally accepted model of W trafficking, with implications for understanding of the mechanisms of NiV immune evasion. - Highlights: • A new model for Nipah virus W protein nucleocytoplasmic trafficking is proposed. • Nipah W protein is shown to undergo active nuclear export via exportin-1. • Nipah W nuclear import is mediated by multiple nuclear localisation signals.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma

γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells and detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

CG13250, a novel bromodomain inhibitor, suppresses proliferation of multiple myeloma cells in an orthotopic mouse model

Multiple myeloma (MM) is characterized by the clonal proliferation of neoplastic plasma cells. Despite a stream of new molecular targets based on better understanding of the disease, MM remains incurable. Epigenomic abnormalities contribute to the pathogenesis of MM. bromodomain 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, binds to acetylated histones during M/G1 transition in the cell cycle promoting progression to S phase. In this study, we investigated the effects of a novel BET inhibitor CG13250 on MM cells. CG13250 inhibited ligand binding to BRD4 in a dose-dependent manner and with an IC{sub 50} value of 1.1 μM. It inhibited MM proliferation in a dose-dependent manner and arrested cells in G1, resulting in the induction of apoptosis through caspase activation. CG13250 inhibited the binding of BRD4 to c-MYC promoter regions suppressing the transcription of the c-MYC gene. Administered in vivo, CG13250 significantly prolonged survival of an orthotopic MM-bearing mice. In conclusion, CG13250 is a novel bromodomain inhibitor that is a promising molecular targeting agent against MM. - Highlights: • A novel bromodomain inhibitor CG13250 suppresses MM cell proliferation. • CG13250 decreases C-MYC expression, resulting in the induction of apoptosis. • CG13250 prolongs the survivals of MM-bearing mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CTP synthase knockdown during early development distorts the nascent vertebral column and causes fluid retention in multiple tissues in zebrafish

Highlights: • Morpholino-based knockdown of ctps during early development produces zebrafish embryos with curved spines and tissue oedema. • Knockdown of ctps1a, and not ctps1b, induces more severe, longer-lasting defects. • Phenotypes are exacerbated when morpholinos against both gene variants are supplied together at uneven ratios. • The same phenotypes were recovered from embryos treated with DON, a CTPS activity-inhibiting drug. CTP Synthase (CTPS) is a metabolic enzyme that is recognized as a catalyst for nucleotide, phospholipid and sialoglycoprotein production. Though the structural characteristics and regulatory mechanisms of CTPS are well-understood, little is known regarding the extent of its involvement during the early developmental stages of vertebrates. Zebrafish carries two CTPS genes, annotated as ctps1a and ctps1b. Phylogenetic analyses show that both genes had diverged from homologues in the ancestral Actinopterygii, Oreochromis niloticus. Conservation of common CTPS-catalytic regions further establishes that both proteins are likely to be functionally similar to hsaCTPS. Here, we show that ctps1a is more critical throughout the initial period of embryonic development than ctps1b. The effects of concurrent partial knockdown are dependent on ctps1a vs ctps1b dosage ratios. When these are equally attenuated, abnormal phenotypes acquired prior to the pharyngula period disappear in hatchlings (48hpf); however, if either gene is more attenuated than the other, these only become more pronounced in advanced stages. Generally, disruption to normal ctps1a or ctps1b expression levels by morpholinos culminates in the distortion of the early spinal column as well as multiple-tissue oedema. Other effects include slower growth rates, increased mortality rates and impaired structural formation of the young fish's extremities. Embryos grown in DON, a glutamine-analogue drug and CTPS antagonist, also exhibit similar characteristics, thus strengthening the validity of the morpholino-induced phenotypes observed. Together, our results demonstrate the importance of CTPS for the development of zebrafish embryos, as well as a disparity in activity and overall importance amongst isozymes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Enhanced antitumor effect of combination of annexin A1 knockdown and bortezomib treatment in multiple myeloma in vitro and in vivo

Highlights: • ANXA1 knockdown suppresses secretion of inflammatory factors in MM cells. • ANXA1 knockdown enhances BTZ-mediated antitumor effect in MM cells in vitro. • ANXA1 knockdown enhances BTZ-mediated antitumor effect in MM cells in vivo. • Combination of ANXA1 silencing and BTZ combination inhibits STAT3 phosphorylation. Bortezomib (BTZ) is one of the most frequently used drugs in treatment of multiple myeloma (MM), but drug-resistance often occurs and limits its clinical efficacy. Annexin A1 (ANXA1) is upregulated in MM, and its knockdown enhances chemosensitivity in MM. However, whether ANXA1 inhibition can increase antitumor activity of BTZ in MM cells remains unknown. In the present study, Cell Counting Kit-8 (CCK-8) and colony formation assays showed that ANXA1 silencing combined with BTZ treatment led to a more significant inhibition of MM cell proliferation than each treatment alone. Cell apoptosis was dramatically promoted in MM cells following silencing of ANXA1 and BTZ administration versus that in ANXA1-silenced alone or BTZ-treated alone cells, as evidenced by decreased expression of phosphorylated signal transducers and activators of transcription 3 and BCL2, and increased expression of BAX. Moreover, we demonstrated that the levels of IL-6 and IL-23 were markedly downregulated in ANXA1-silenced and BTZ-treated MM cells. Furthermore, the combination of ANXA1 knockdown and BTZ treatment distinctly suppressed tumor growth in vivo compared with BTZ treatment alone. Taken together, our results show that downregulation of ANXA1 enhances antitumor activity of BTZ in MM in vitro and in vivo, indicating that ANXA1 may be a promising target for enhancing the chemosensitivity of MM to BTZ.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Chemical-free production of multiple high-value bioproducts from metabolically engineered transgenic sugarcane ‘oilcane’ bagasse and their recovery using nanofiltration

Bioenergy crops have potential for being a sustainable and renewable feedstock for biofuels and various value- added bioproducts. The study utilizes recently developed transgenic sugarcane (‘oilcane’) bagasse for chemical- free coproduction of high-value bioproducts, i.e., furfurals, HMF, acetic acid, cellulosic sugars, and vegetative lipids. Hydrothermal pretreatment was optimized at 210°C for 5 min to coproduce 6.91%, 2.67%, 5.07%, 2.42% and 37.82% (w/w) furfurals, HMF, acetic acid, vegetative lipids, and cellulosic sugars, respectively from lignocellulosic biomass. Additionally, nanofiltration system in-series was successfully established to recover sugars, furfurals, HMF, and acetic acid from the pretreatment liquor. 1st nanofiltration with Duracid NF mem- brane rejected ~99% sugars. Concentrated sugars with significantly reduced inhibitory products were obtained in retentate for fermentation. 2nd nanofiltration with NF90 membrane used permeate of 1st nanofiltration as feed and rejected ~ 86% furfurals. Furthermore, the work demonstrates the feasibility of coproducing and recovering multiple biochemicals from lignocellulosic biomass.

09 BIOMASS FUELS↗
DOE OSTI2016

Multiple CaMKII Binding Modes to the Actin Cytoskeleton Revealed by Single-Molecule Imaging

Localization of the Ca2þ/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIa (a) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIb (b). The bE0 splice variant bound more weakly than a, showing that binding by b depends critically on the interdomain linker. The mutations bT287D and aT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (bK43R and aK42M) or Ca2þ/calmodulin (bA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the aT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIa isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.

59 BASIC BIOLOGICAL SCIENCES↗