Real-time single-molecule imaging of CaMKII-calmodulin interactions

Although the recent identification of the mitochondrial Ca2+ uniporter (MCU) has resolved a long-standing mystery as to how Ca2+ freely enters the mitochondria, it has also evoked additional questions such as its mode of regulation and the identity of other associated factors. In an article recently published in Nature , Joiner et al1 provide data demonstrating that in the heart, matrix-localized Ca2+/calmodulin-dependent protein kinase II (CaMKII) can upregulate MCU activity in a manner requiring phosphorylation of the channel N terminus. They showed that inhibition of CaMKII-dependent MCU activity protected the heart from ischemic injury by presumably reducing Ca2+ influx and desensitizing the mitochondrial permeability transition pore (MPTP) to opening. Although these results demonstrate convincingly that CaMKII plays an important role in MCU regulation and subsequent response to cardiac injury, several questions remain unanswered. The ability of mitochondria to take up and sequester Ca2+ plays an important role in the buffering of cytosolic Ca2+, regulation of ATP production via the citric acid cycle, and regulation of apoptotic and necrotic cell death pathways.2 Although mitochondrial Ca2+ uptake was first described in the 1960s3 and the electrophysiological properties of the MCU were reported in 2004,4 it was not until 2011 that 2 articles were published revealing the genetic identity of the MCU.5,6 This pioneering work has initiated a search for additional members of the MCU complex (such as MICU17 and the recently discovered MCUR1),8 as well as an attempt to understand how MCU-mediated Ca2+ influx participates in the regulation of whole-cell Ca2+ signaling and whether well-described pathways that regulate other Ca2+ handling processes can similarly modulate MCU-dependent Ca2+ uptake. In the featured article, Joiner et al1 show that CaMKII serves …

Exposure to psychostimulant drugs leads to sensitized locomotor responding, nucleus accumbens (NAcc) dopamine (DA) overflow, and drug self-administration upon reexposure to the drug weeks to months later. Calcium-dependent signaling pathways contribute importantly to both the induction and expression of sensitization by these drugs. In particular, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII), a serine/threonine kinase that is highly expressed in forebrain regions such as the NAcc, is known to contribute to the expression of several forms of plasticity including sensitization. Notably, pharmacologically inhibiting CaMKII in the NAcc prevents the expression of sensitized locomotion, NAcc DA overflow, and drug taking. Evidence indicates that CaMKII may act both pre- and postsynaptically in this site to influence the expression of these manifestations of sensitization. Presynaptically in DA neuron terminals, CaMKII regulates sensitized DA overflow. Postsynaptically in medium spiny neurons, it contributes to the upregulation of AMPA receptors, the activation of which is necessary for the expression of sensitization. It is conceivable that interactions between CaMKII-mediated neuroadaptations in both of these sites may underlie the long-lasting maintenance of sensitization by psychostimulant drugs.

InsP(3)-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP(3)R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P(o)). We have identified S150 in the InsP(3)R2 core suppressor domain (amino acids 1-225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP(3)R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P(o) under normal recording conditions in the absence of CaMKII (2 μM InsP(3) and 250 nM [Ca(2+)](FREE)) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP(3)R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP(3)R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.

The human pregnane X receptor (hPXR) is recognized as a xenobiotic-sensing nuclear receptor that transcriptionally regulates the gene expression of drug-metabolizing enzymes and transporters. Our study elucidates the mechanism by which the localization of hPXR is regulated through threonine-290. A phosphomimetic mutation at threonine-290 (T290D) retained hPXR in the cytoplasm of HepG2, HuH6, and SW480 cells in vitro and the mouse liver in vivo even after treatment with rifampicin, and a phosphodeficient mutation (T290A) translocated from the cytoplasm to the nucleus as the wild-type hPXR. The amount of the unphosphorylated wild-type yellow fluorescent protein-hPXR fusion protein but not the T290A mutant increased on Phos-tag gels in response to stimulations with rifampicin and cyclin-dependent kinase 2 inhibitor roscovitine, and a marked increase was observed in the unphosphorylated levels of the T290A mutant in nontreated cells. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)] and transfection with anti-CaMKII small-interfering RNA (siRNA) enhanced the unphosphorylated levels of the wild-type protein. CaMKII directly phosphorylated the threonine-290 of hPXR, and the T290A mutant conferred resistance to CaMKII. The protein phosphatase (PP) inhibitor okadaic acid (100 nM) and transfection with anti-PP1 siRNA but not anti-PP2A siRNA led to reduced expression of CYP3A4 mRNA. After the rifampicin and roscovitine stimulations, PP1 was recruited to the wild-type hPXR but not the T290A mutant. These results suggest that phosphorylation at threonine-290 by CaMKII may impair the function of hPXR by repressing its translocation to the nucleus, and dephosphorylation by PP1 is necessary for the xenobiotic-dependent nuclear translocation of hPXR.

Acute Myeloid Leukemia (AML) is the most common form of leukemia diagnosed in adults with the lowest survival rates. It is a heterogeneous disease and incidence rates increase yearly, with the overall survival of AML patients remaining low. Emerging treatment strategies are key in improving the recovery rates of those diagnosed with AML due to not all patients responding to currently used intense chemotherapeutics. For the best treatment of AML patients possible, new treatments must be developed and treatment biomarkers identified. Tetrandrine is a calcium channel blocker that is a potential treatment. It has demonstrated anti-proliferation, pro-autophagic and apoptotic, and multidrug resistance reversal effects on solid cancer varieties, thought to be potentiated by acquired genomic abnormalities, and it is hypothesised that it could be repurposed to act in a similar role in haematological cancers, namely AML. Although research into the effects of tetrandrine in AML is limited, the genomic abnormalities within subsets of the disease may reveal patients that would benefit from tetrandrine treatment. This review aims to explore the potential mechanisms of tetrandrine treatment in AML, drawing on information gained through tetrandrine treatment in solid cancer. The main focus will be interactions with genes, protein, and pathways commonly dysregulated in AML, namely Tumour Protein 53 (TP53), C-Myc (MYC), Kirsten Rate Sarcoma Virus (KRAS), Vascular Endothelial Growth Factor (VEGF), Fms-like tyrosine kinase 3 (FLT3) and Nuclear Receptor Subfamily 4A1 (NR4A1) as well as the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)-inflammasome and calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII).

Voltage-gated calcium channels (VGCCs), calmodulin (CaM), and calmodulin kinase II (CaMKII) are essential for various nervous system functions. CaM and CaMKII differentially regulate calcium dependent facilitation (CDF) and calcium dependent inactivation (CDI) of the Cav1 and Cav2 families of VGCCs. It is generally accepted that conserved structures in the C-terminus of these channels regulate CDF and CDI, and yet recent evidence indicates that other intracellular regions may be involved. We recently discovered that N-terminal sequences in Cav1.2 bind CaM and CaMKII, and function to regulate CDI as well as surface expression and open probability, respectively. Cav1 and Cav2 share significant portions of N-terminal sequence and therefore we explored whether homologous binding sites might exist in Cav2.1. Here, we show that like the proximal N-terminus of Cav1.2, the homologous region of Cav2.1 contains sequences which interact either directly or indirectly with CaM.

In phagocytes, activation of the respiratory burst by chemoattractants requires ATP and involves a pertussis toxin-sensitive G protein. ATP is also required for the response elicited in permeabilized neutrophils by nonhydrolyzable GTP analogs, indicating that at least one of the ATP-dependent steps lies downstream of the receptor-coupled G protein(s). A respiratory burst can also be produced in a reconstituted cell-free system by addition of arachidonic acid. Most investigators find this response to be independent of ATP, yet stimulated by GTP analogs, implying that the ATP-dependent steps observed in the unbroken cells must precede the guanine nucleotide-requiring event. To resolve this apparent discrepancy, we studied the ATP and guanine nucleotide dependence of the oxidative response elicited by arachidonic acid in electrically permeabilized human neutrophils. Two components of the response were apparent: one was ATP-dependent, the other ATP-independent. The ATP-dependent component was partially inhibited by staurosporine, suggesting involvement of protein kinase C. This kinase signals activation of the NADPH oxidase without intervening G proteins, since stimulation by phorbol ester was unaffected by guanosine 5'-(beta-thio)diphosphate (GDP beta S). Although nonhydrolyzable GTP analogs failed to stimulate the oxidase in the absence of ATP, the ATP-independent response stimulated by arachidonic acid was found to require GTP or one of its analogs and to be inhibited by GDP beta S. The relative potency of the guanine nucleotides to support the arachidonic acid response in the absence of ATP (5'-guanylyl imidodiphosphate (GMP-PNP) greater than or equal to guanosine 5'-(gamma-thio)triphosphate GTP gamma S) greater than or equal to (GTP) differed from their efficacy to stimulate the burst in the presence of ATP (GTP gamma S greater than GMP-PNP much greater than GTP). These observations suggest the involvement of two distinct GTP-binding proteins in oxidase activation: a receptor-coupled, heterotrimeric, pertussis toxin-sensitive G protein, and a second GTP-binding protein(s) located downstream of the ATP-requiring steps, which may lie in close proximity to the NADPH oxidase. This secondary GTP-binding protein could be part of the pathway activated by chemoattractants, but does not mediate stimulation via protein kinase C. Therefore multiple parallel routes may exist for activation of the NADPH oxidase.

Phosphorylation of syntaxin 3B by CaMKII regulates the formation of t-SNARE complexes

Lipidated gemini peptide amphiphiles with enhanced loading capacity and cell membrane affinity for drug delivery

Live-cell single-molecule imaging reveals clathrin and caveolin-1 dependent docking of SMAD4 at the cell membrane

High-throughput single-molecule total internal reflection fluorescence microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous-fused silica interface. At high pH and low ionic strength, negatively charged BSA adsorbed slowly to the negatively charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range nonelectrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g., adsorption and desorption) under steady-state conditions, which would be impossible to measure using ensemble-averaging methods.

The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms.

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

Insights into the Autoinhibition Mechanism of the Tiam1 Guanine Nucleotide Exchange Factor

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.