Phosphorylation of PSD-95 at serine 73 in dCA1 is required for extinction of contextual fear.

The updating of contextual memories is essential for survival in a changing environment. Accumulating data indicate that the dorsal CA1 area (dCA1) contributes to this process. However, the cellular and molecular mechanisms of contextual fear memory updating remain poorly understood. Postsynaptic density protein 95 (PSD-95) regulates the structure and function of glutamatergic synapses. Here, using dCA1-targeted genetic manipulations in vivo, combined with ex vivo 3D electron microscopy and electrophysiology, we identify a novel, synaptic mechanism that is induced during attenuation of contextual fear memories and involves phosphorylation of PSD-95 at Serine 73 in dCA1. Our data provide the proof that PSD-95-dependent synaptic plasticity in dCA1 is required for updating of contextual fear memory.

SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

ATM phosphorylates PP2A subunit A resulting in nuclear export and spatiotemporal regulation of the DNA damage response.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

Autophosphorylation of αCaMKII regulates alcohol consumption by controlling sedative effects of alcohol and alcohol-induced loss of excitatory synapses.

Calcium/calmodulin-dependent kinase II (CaMKII) is a key enzyme at the glutamatergic synapses. CAMK2A gene variants have been linked with alcohol use disorder (AUD) by an unknown mechanism. Here, we looked for the link between αCaMKII autophosphorylation and the AUD aetiology. Autophosphorylation-deficient heterozygous αCaMKII mutant mice (T286A+/- ) were trained in the IntelliCages to test the role of αCaMKII activity in AUD-related behaviours. The glutamatergic synapses morphology in CeA was studied in the animals drinking alcohol using 3D electron microscopy. We found that T286A+/- mutants consumed less alcohol and were more sensitive to sedating effects of alcohol, as compared to wild-type littermates (WT). After voluntary alcohol drinking, T286A+/- mice had less excitatory synapses in the CeA, as compared to alcohol-naive animals. This change correlated with alcohol consumption was not reversed after alcohol withdrawal and not observed in WT mice. Our study suggests that αCaMKII autophosphorylation affects alcohol consumption by controlling sedative effects of alcohol and preventing synaptic loss in the individuals drinking alcohol. This finding advances our understanding of the molecular processes that regulate alcohol dependence.

PSD-95 in CA1 Area Regulates Spatial Choice Depending on Age.

Cognitive processes that require spatial information rely on synaptic plasticity in the dorsal CA1 area (dCA1) of the hippocampus. Since the function of the hippocampus is impaired in aged individuals, it remains unknown how aged animals make spatial choices. Here, we used IntelliCage to study behavioral processes that support spatial choices of aged female mice living in a group. As a proxy of training-induced synaptic plasticity, we analyzed the morphology of dendritic spines and the expression of a synaptic scaffold protein, PSD-95. We observed that spatial choice training in young adult mice induced correlated shrinkage of dendritic spines and downregulation of PSD-95 in dCA1. Moreover, long-term depletion of PSD-95 by shRNA in dCA1 limited correct choices to a reward corner, while reward preference was intact. In contrast, old mice used behavioral strategies characterized by an increased tendency for perseverative visits and social interactions. This strategy resulted in a robust preference for the reward corner during the spatial choice task. Moreover, training decreased the correlation between PSD-95 expression and the size of dendritic spines. Furthermore, PSD-95 depletion did not impair place choice or reward preference in old mice. Thus, our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices, old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment.SIGNIFICANCE STATEMENT It remains poorly understood how aging affects behavioral and molecular processes that support cognitive functions. It is, however, essential to understand these processes to develop therapeutic interventions that support successful cognitive aging. Our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices (i.e., choices that require spatial information), old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment. Second, the contribution of PSD-95-dependent synaptic functions in spatial choice changes with age.

Delayed KCNQ1/KCNE1 assembly on the cell surface helps IKs fulfil its function as a repolarization reserve in the heart.

KEY POINTS: In adult ventricular myocytes, the slow delayed rectifier (IKs ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. ABSTRACT: Slow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during ß-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to ß-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.

Long-term Memory Upscales Volume of Postsynaptic Densities in the Process that Requires Autophosphorylation of αCaMKII.

It is generally accepted that formation and storage of memory relies on alterations of the structure and function of brain circuits. However, the structural data, which show learning-induced and long-lasting remodeling of synapses, are still very sparse. Here, we reconstruct 1927 dendritic spines and their postsynaptic densities (PSDs), representing a postsynaptic part of the glutamatergic synapse, in the hippocampal area CA1 of the mice that underwent spatial training. We observe that in young adult (5 months), mice volume of PSDs, but not the volume of the spines, is increased 26 h after the training. The training-induced growth of PSDs is specific for the dendritic spines that lack smooth endoplasmic reticulum and spine apparatuses, and requires autophosphorylation of αCaMKII. Interestingly, aging alters training-induced ultrastructural remodeling of dendritic spines. In old mice, both the median volumes of dendritic spines and PSDs shift after training toward bigger values. Overall, our data support the hypothesis that formation of memory leaves long-lasting footprint on the ultrastructure of brain circuits; however, the form of circuit remodeling changes with age.

Dynamic palmitoylation regulates trafficking of K channel interacting protein 2 (KChIP2) across multiple subcellular compartments in cardiac myocytes.

BACKGROUND: K channel interacting protein 2 (KChIP2), initially cloned as Kv4 channel modulator, is a multi-tasking protein. In addition to modulating several cardiac ion channels at the plasma membrane, it can also modulate microRNA transcription inside nuclei, and interact with presenilins to modulate Ca release through RyR2 in the cytoplasm. However, the mechanism regulating its subcellular distribution is not clear. OBJECTIVE: We tested whether palmitoylation drives KChIP2 trafficking and distribution in cells, and whether the distribution pattern of KChIP2 in cardiac myocytes is sensitive to cellular milieu. METHOD: We conducted imaging and biochemical experiments on palmitoylatable and unpalmitoylatable KChIP2 variants expressed in COS-7 cells and in cardiomyocytes, and on native KChIP2 in myocytes. RESULTS: In COS-7 cells, palmitoylatable KChIP2 clustered to plasma membrane, while unpalmitoylatable KChIP2 exhibited higher cytoplasmic mobility and faster nuclear entry. The same differences in distribution and mobility were observed when these KChIP2 variants were expressed in cardiac myocytes, indicating that the palmitoylation-dependent distribution and trafficking are intrinsic properties of KChIP2. Importantly, acute stress in a rat model of cardiac arrest/resuscitation induced changes in native KChIP2 resembling those of KChIP2 depalmitoylation, promoting KChIP2 nuclear entry. CONCLUSION: The palmitoylation status of KChIP2 determines its subcellular distribution in cardiac myocytes. Stress promotes nuclear entry of KChIP2, diverting it from ion channel modulation at the plasma membrane to other functions in the nuclear compartment.

Quality of biological images, reconstructed using localization microscopy data.

Motivation: Fluorescence localization microscopy is extensively used to study the details of spatial architecture of subcellular compartments. This modality relies on determination of spatial positions of fluorophores, labeling an extended biological structure, with precision exceeding the diffraction limit. Several established models describe influence of pixel size, signal-to-noise ratio and optical resolution on the localization precision. The labeling density has been also recognized as important factor affecting reconstruction fidelity of the imaged biological structure. However, quantitative data on combined influence of sampling and localization errors on the fidelity of reconstruction are scarce. It should be noted that processing localization microscopy data is similar to reconstruction of a continuous (extended) non-periodic signal from a non-uniform, noisy point samples. In two dimensions the problem may be formulated within the framework of matrix completion. However, no systematic approach has been adopted in microscopy, where images are typically rendered by representing localized molecules with Gaussian distributions (widths determined by localization precision). Results: We analyze the process of two-dimensional reconstruction of extended biological structures as a function of the density of registered emitters, localization precision and the area occupied by the rendered localized molecule. We quantify overall reconstruction fidelity with different established image similarity measures. Furthermore, we analyze the recovered similarity measure in the frequency space for different reconstruction protocols. We compare the cut-off frequency to the limiting sampling frequency, as determined by labeling density. Availability and implementation: The source code used in the simulations along with test images is available at https://github.com/blazi13/qbioimages. Contact: bruszczy@nencki.gov.pl or t.bernas@nencki.gov.pl. Supplementary information: Supplementary data are available at Bioinformatics online.

Mitochondria Targeting with Luminescent Rhenium(I) Complexes.

Two new neutral fac-[Re(CO)3(phen)L] compounds (1,2), with phen = 1,10-phenanthroline and L = O2C(CH2)5CH3 or O2C(CH2)4C≡CH, were synthetized in one-pot procedures from fac-[Re(CO)3(phen)Cl] and the corresponding carboxylic acids, and were fully characterized by IR and UV-Vis absorption spectroscopy, ¹H- and 13C-NMR, mass spectrometry and X-ray crystallography. The compounds, which display orange luminescence, were used as probes for living cancer HeLa cell staining. Confocal microscopy revealed accumulation of both dyes in mitochondria. To investigate the mechanism of mitochondrial staining, a new non-emissive compound, fac-[Re(CO)3(phen)L], with L = O2C(CH2)3((C5H5)Fe(C5H4), i.e., containing a ferrocenyl moiety, was synthetized and characterized (3). 3 shows the same mitochondrial accumulation pattern as 1 and 2. Emission of 3 can only be possible when ferrocene-containing ligand dissociates from the metal center to produce a species containing the luminescent fac-[Re(CO)3(phen)]⁺ core. The release of ligands from the Re center was verified in vitro through the conjugation with model proteins. These findings suggest that the mitochondria accumulation of compounds 1-3 is due to the formation of luminescent fac-[Re(CO)3(phen)]⁺ products, which react with cellular matrix molecules giving secondary products and are uptaken into the negatively charged mitochondrial membranes. Thus, reported compounds feature a rare dissociation-driven mechanism of action with great potential for biological applications.

Pyrene-nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies.

Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene-C(O)CH2CH2-thymine (2) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base-base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA)10-T10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells.

Single-cell imaging of the heat-shock response in colon cancer cells suggests that magnitude and length rather than time of onset determines resistance to apoptosis.

Targeting the proteasome is a valuable approach for cancer therapy, potentially limited by pro-survival pathways that are induced in parallel to cell death. Whether these pro-survival pathways are activated in all cells, show different activation kinetics in sensitive versus resistant cells or interact functionally with cell death pathways is unknown. We monitored activation of the heat-shock response (HSR), a key survival pathway induced by proteasome inhibition, relative to apoptosis activation in HCT116 colon cancer cells expressing enhanced green fluorescent protein (EGFP) under the control of the HSP70 promoter. Single-cell and high-content time-lapse imaging of epoxomicin treatment revealed that neither basal activity nor the time of onset of the HSR differed between resistant and sensitive populations. However, resistant cells had significantly higher and prolonged reporter activity than those that succumbed to cell death. p53 deficiency protected against cell death but failed to modulate the HSR. By contrast, inhibition of the HSR significantly increased the cytotoxicity of epoxomicin. Our data provide novel insights into the kinetics and heterogeneity of the HSR during proteasome inhibition, suggesting that the HSR modulates cell death signalling unidirectionally.

Inhibition of SCD1 impairs palmitate-derived autophagy at the step of autophagosome-lysosome fusion in pancreatic ß-cells.

Autophagy is indispensable for the proper architecture and flawless functioning of pancreatic ß-cells. A growing body of evidence indicates reciprocal communication between autophagic pathways, apoptosis, and intracellular lipids. The way in which elevated levels of free saturated or unsaturated FAs contribute to progressive ß-cell failure remains incompletely understood. Stearoyl-CoA desaturase (SCD)1, a key regulatory enzyme in biosynthesis of MUFAs, was shown to play an important role in regulation of ß-cell function. Here, we investigated whether SCD1 activity is engaged in palmitate-induced pancreatic ß-cell autophagy. We found augmented apoptosis and diminished autophagy upon cotreatment of INS-1E cells with palmitate and an SCD1 inhibitor. Furthermore, we found that additional treatment of the cells with monensin, an inhibitor of autophagy at the step of fusion, exacerbates palmitate-induced apoptosis. Accordingly, diminished SCD1 activity affected the accumulation, composition, and saturation status of cellular membrane phospholipids and neutral lipids. Such an effect was accompanied by aberrant endoplasmic reticulum stress, mitochondrial injury, and decreases in insulin secretion and cell proliferation. Our data reveal a novel mechanism by which the inhibition of SCD1 activity affects autophagosome-lysosome fusion because of perturbations in cellular membrane integrity, thus leading to an aberrant stress response and ß-cell failure.

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-κB (NF-κB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-κB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-κB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-κB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-κB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-κB activity in neurons.

Persistent PKA activation redistributes NaV1.5 to the cell surface of adult rat ventricular myocytes.

During chronic stress, persistent activation of cAMP-dependent protein kinase (PKA) occurs, which can contribute to protective or maladaptive changes in the heart. We sought to understand the effect of persistent PKA activation on NaV1.5 channel distribution and function in cardiomyocytes using adult rat ventricular myocytes as the main model. PKA activation with 8CPT-cAMP and okadaic acid (phosphatase inhibitor) caused an increase in Na+ current amplitude without altering the total NaV1.5 protein level, suggesting a redistribution of NaV1.5 to the myocytes' surface. Biotinylation experiments in HEK293 cells showed that inhibiting protein trafficking from intracellular compartments to the plasma membrane prevented the PKA-induced increase in cell surface NaV1.5. Additionally, PKA activation induced a time-dependent increase in microtubule plus-end binding protein 1 (EB1) and clustering of EB1 at myocytes' peripheral surface and intercalated discs (ICDs). This was accompanied by a decrease in stable interfibrillar microtubules but an increase in dynamic microtubules along the myocyte surface. Imaging and coimmunoprecipitation experiments revealed that NaV1.5 interacted with EB1 and ß-tubulin, and both interactions were enhanced by PKA activation. We propose that persistent PKA activation promotes NaV1.5 trafficking to the peripheral surface of myocytes and ICDs by providing dynamic microtubule tracks and enhanced guidance by EB1. Our proposal is consistent with an increase in the correlative distribution of NaV1.5, EB1, and ß-tubulin at these subcellular domains in PKA-activated myocytes. Our study suggests that persistent PKA activation, at least during the initial phase, can protect impulse propagation in a chronically stressed heart by increasing NaV1.5 at ICDs.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.

Stearoyl-CoA desaturase 1 deficiency exacerbates palmitate-induced lipotoxicity by the formation of small lipid droplets in pancreatic ß-cells.

The accelerating accumulation of surplus lipids in the pancreas triggers structural and functional changes in type 2 diabetes-affected islets. Pancreatic ß-cells exhibit a restricted capacity to store fat reservoirs in lipid droplets (LDs), which act as transient buffers to prevent lipotoxic stress. With the increasing incidence of obesity, growing interest has been seen in the intracellular regulation of LD metabolism for ß-cell function. Stearoyl-CoA desaturase 1 (SCD1) is critical for producing unsaturated fatty acyl moieties for fluent storage into and out of LDs, likely affecting the overall rate of ß-cell survival. We explored LD-associated composition and remodeling in SCD1-deprived INS-1E cells and in pancreatic islets in wildtype and SCD1-/- mice in the lipotoxic milieu. Deficiency in the enzymatic activity of SCD1 led to decrease in the size and number of LDs and the lower accumulation of neutral lipids. This occurred in parallel with a higher compactness and lipid order inside LDs, followed by changes in the saturation status and composition of fatty acids within core lipids and the phospholipid coat. The lipidome of LDs was enriched in 18:2n-6 and 20:4n-6 in ß-cells and pancreatic islets. These rearrangements markedly contributed to differences in protein association with the LD surface. Our findings highlight an unexpected molecular mechanism by which SCD1 activity affects the morphology, composition and metabolism of LDs. We demonstrate that SCD1-dependent disturbances in LD enrichment can impact pancreatic ß-cells and islet susceptibility to palmitate, which may have considerable diagnostic and methodological value for the characterization of LDs in human ß-cells in type 2 diabetes patients.