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Mitochondria are small cellular constituents that generate cellular energy (ATP) by oxidative phosphorylation (OXPHOS). Dysfunction of these organelles is linked to a heterogeneous group of multisystemic disorders, including diabetes, cancer, ageing-related pathologies and rare mitochondrial diseases. With respect to the latter, mutations in subunit-encoding genes and assembly factors of the first OXPHOS complex (complex I) induce isolated complex I deficiency and Leigh syndrome. This syndrome is an early-onset, often fatal, encephalopathy with a variable clinical presentation and poor prognosis due to the lack of effective intervention strategies. Mutations in the nuclear DNA-encoded NDUFS4 gene, encoding the NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4) of complex I, induce 'mitochondrial complex I deficiency, nuclear type 1' (MC1DN1) and Leigh syndrome in paediatric patients. A variety of (tissue-specific) Ndufs4 knockout mouse models were developed to study the Leigh syndrome pathomechanism and intervention testing. Here, we review and discuss the role of complex I and NDUFS4 mutations in human mitochondrial disease, and review how the analysis of Ndufs4 knockout mouse models has generated new insights into the MC1ND1/Leigh syndrome pathomechanism and its therapeutic targeting. [ABSTRACT FROM AUTHOR]

Background: Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideally, co-expression occurs at a defined ratio, which is constant among cells. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Förster Resonance Energy Transfer (FRET) sensors. Methodology/Principal Findings: Four co-expression strategies based on co-transfection, a dual promotor plasmid, an internal ribosome entry site (IRES) and a viral 2A peptide were selected. Co-expression of two spectrally separable fluorescent proteins in single living cells was quantified. It is demonstrated that the 2A peptide strategy can be used for robust equimolar co-expression, while the IRES sequence allows expression of two proteins at a ratio of approximately 3:1. Combined 2A and IRES elements were used for the construction of a single plasmid that drives expression of three individual proteins, which generates a FRET sensor for measuring heterotrimeric G-protein activation. The plasmid drives co-expression of donor and acceptor tagged subunits, with reduced heterogeneity, and can be used to measure G-protein activation in single living cells. Conclusions/Significance: Quantitative co-expression of two or more proteins can be achieved with little cell-to-cell variability. This finding enables reliable co-expression of donor and acceptor tagged proteins for FRET studies, which is of particular importance for the development of novel bimolecular sensors that can be expressed from single plasmid. [ABSTRACT FROM AUTHOR]

Background: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. Results: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. Conclusions: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity. [ABSTRACT FROM AUTHOR]

Paediatric Leigh syndrome (LS) is an early-onset and fatal neurodegenerative disorder lacking treatment options. LS is frequently caused by mutations in the NDUFS4 gene, encoding an accessory subunit of mitochondrial complex I (CI), the first complex of the oxidative phosphorylation (OXPHOS) system. Whole-body Ndufs4 knockout (KO) mice (WB-KO mice) are widely used to study isolated CI deficiency, LS pathology and interventions. These animals develop a brain-specific phenotype via an incompletely understood pathomechanism. Here we performed a quantitative analysis of the sub-brain proteome in six-weeks old WB-KO mice vs. wildtype mice. Brain regions comprised of a brain slice (BrSl), cerebellum (CB), cerebral cortex (CC), hippocampus (HC), inferior colliculus (IC), and superior colliculus (SC). Proteome analysis demonstrated similarities between CC/HC, and between IC/SC, whereas BrSl and CB differed from these two groups and each other. All brain regions displayed greatly reduced levels of two CI structural subunits (NDUFS4, NDUFA12) and an increased level of the CI assembly factor NDUFAF2. The level of CI-Q module subunits was significantly more reduced in IC/SC than in BrSl/CB/CC/HC, whereas other OXPHOS complex levels were not reduced. Gene ontology and pathway analysis demonstrated specific and common proteome changes between brain regions. Across brain regions, upregulation of cold-shock-associated proteins, mitochondrial fatty acid (FA) oxidation and synthesis (mtFAS) were the most prominent. FA-related pathways were predominantly upregulated in CB and HC. Based upon these results, we argue that stimulation of these pathways is futile and pro-pathological and discuss alternative strategies for therapeutic intervention in LS. SIGNIFICANCE: The Ndufs4 knockout mouse model is currently the most relevant and most widely used animal model to study the brain-linked pathophysiology of human Leigh Syndrome (LS) and intervention strategies. We demonstrate that the Ndufs4 knockout brain engages futile and pro-pathological responses. These responses explain both negative and positive outcomes of intervention studies in Leigh Syndrome mice and patients, thereby guiding novel intervention opportunities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Hydroxyapatite nanoparticles (nHA) have gained attention as potential intracellular drug delivery vehicles due to their high binding affinity for various biomolecules and pH-dependent solubility. Yet, the dependence of nHA cytocompatibility on their physicochemical properties remains unclear since numerous studies have revealed starkly contrasting results. These discrepancies may be attributed to differences in size, shape, crystallinity, and aggregation state of nHA, which complicates fundamental understanding of the factors driving nHA cytotoxicity. Here, we hypothesize that nHA cytotoxicity is primarily driven by intracellular calcium levels following the internalization of nHA nanoparticles. By investigating the cytotoxicity of spherical nHA with different crystallinity and dispersity, we find that both lower crystallinity and increased agglomeration of nHA raise cytotoxicity, with nanoparticle agglomeration being the more dominant factor. We show that the internalization of nHA enhances intracellular calcium levels and increases the production of reactive oxygen species (ROS). However, only subtle changes in intracellular calcium are observed, and their physiological relevance remains to be confirmed. In conclusion, we show that nHA agglomeration enhances ROS production and the associated cytotoxicity. These findings provide important guidelines for the future design of nHA-containing formulations for biomedical applications, implying that nHA crystallinity and especially agglomeration should be carefully controlled to optimize biocompatibility and therapeutic efficacy.

Mitochondrial membrane potential (Δψ) and morphology are considered key readouts of mitochondrial functional state. This morphofunction can be studied using fluorescent dyes ("probes") like tetramethylrhodamine methyl ester (TMRM) and Mitotrackers (MTs). Although these dyes are broadly used, information comparing their performance in mitochondrial morphology quantification and Δψ-sensitivity in the same cell model is still scarce. Here we applied epifluorescence microscopy of primary human skin fibroblasts to evaluate TMRM, Mitotracker Red CMXros (CMXros), Mitotracker Red CMH 2 Xros (CMH2Xros), Mitotracker Green FM (MG) and Mitotracker Deep Red FM (MDR). All probes were suited for automated quantification of mitochondrial morphology parameters when Δψ was normal, although they did not deliver quantitatively identical results. The mitochondrial localization of TMRM and MTs was differentially sensitive to carbonyl cyanide-4-phenylhydrazone (FCCP)-induced Δψ depolarization, decreasing in the order: TMRM ≫ CHM2Xros = CMXros = MDR > MG. To study the effect of reversible Δψ changes, the impact of photo-induced Δψ "flickering" was studied in cells co-stained with TMRM and MG. During a flickering event, individual mitochondria displayed subsequent TMRM release and uptake, whereas this phenomenon was not observed for MG. Spatiotemporal and computational analysis of the flickering event provided evidence that TMRM redistributes between adjacent mitochondria by a mechanism dependent on Δψ and TMRM concentration. In summary, this study demonstrates that: (1) TMRM and MTs are suited for automated mitochondrial morphology quantification, (2) numerical data obtained with different probes is not identical, and (3) all probes are sensitive to FCCP-induced Δψ depolarization, with TMRM and MG displaying the highest and lowest sensitivity, respectively. We conclude that TMRM is better suited for integrated analysis of Δψ and mitochondrial morphology than the tested MTs under conditions that Δψ is not substantially depolarized., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: WJHK is an ad-hoc scientific advisor of Khondrion B.V. (Nijmegen, The Netherlands). This SME had no involvement in the data collection, analysis and interpretation, writing of the manuscript, and in the decision to submit the manuscript for publication., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ionic calcium (Ca 2+ ) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca 2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca 2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca 2+ uptake., Competing Interests: Declaration of competing interest WJHK is a scientific advisor of Khondrion B.V. (Nijmegen, The Netherlands). This company was not involved in the data analysis and interpretation, writing of the manuscript, and in the decision to submit the manuscript for publication., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP + ) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C 10 -TPP + ). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are small cellular constituents that generate cellular energy (ATP) by oxidative phosphorylation (OXPHOS). Dysfunction of these organelles is linked to a heterogeneous group of multisystemic disorders, including diabetes, cancer, ageing-related pathologies and rare mitochondrial diseases. With respect to the latter, mutations in subunit-encoding genes and assembly factors of the first OXPHOS complex (complex I) induce isolated complex I deficiency and Leigh syndrome. This syndrome is an early-onset, often fatal, encephalopathy with a variable clinical presentation and poor prognosis due to the lack of effective intervention strategies. Mutations in the nuclear DNA-encoded NDUFS4 gene, encoding the NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4) of complex I, induce 'mitochondrial complex I deficiency, nuclear type 1' (MC1DN1) and Leigh syndrome in paediatric patients. A variety of (tissue-specific) Ndufs4 knockout mouse models were developed to study the Leigh syndrome pathomechanism and intervention testing. Here, we review and discuss the role of complex I and NDUFS4 mutations in human mitochondrial disease, and review how the analysis of Ndufs4 knockout mouse models has generated new insights into the MC1ND1/Leigh syndrome pathomechanism and its therapeutic targeting., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Classical mitochondrial disease (MD) represents a group of complex metabolic syndromes primarily linked to dysfunction of the mitochondrial ATP-generating oxidative phosphorylation (OXPHOS) system. To date, effective therapies for these diseases are lacking. Here we discuss the ketogenic diet (KD), being a high-fat, moderate protein, and low carbohydrate diet, as a potential intervention strategy. We concisely review the impact of the KD on bioenergetics, ROS/redox metabolism, mitochondrial dynamics and mitophagy. Next, the consequences of the KD in (models of) MD, as well as KD adverse effects, are described. It is concluded that the current experimental evidence suggests that the KD can positively impact on mitochondrial bioenergetics, mitochondrial ROS/redox metabolism and mitochondrial dynamics. However, more information is required on the bioenergetic consequences and mechanistic mode-of-action aspects of the KD at the cellular level and in MD patients., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4 -/- mouse tissues. Ndufs4 -/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4 -/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4 -/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4 -/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

At concentrations exceeding 10 μM, arginine-rich cell-penetrating peptides (CPPs) trigger a rapid cytoplasmic import that involves activation of acid sphingomyelinase (ASMase). ASMase activation occurs through a variety of stress signals and has also been related to the reorganization of membrane microdomains during entry of pathogens. However, in none of these cases has the initial trigger for ASMase activation been established on a molecular level. We here show that rapid cytosolic CPP import depends upon an increase in intracellular calcium, likely caused by modulation of the Orai1 calcium channel. At low peptide concentration, cytoplasmic import could be induced by thapsigargin, a known activator of Orai1. Compounds known to block Orai1 inhibited rapid uptake. Peptide-mediated modulation of Orai1 involved cell surface sialic acids as inhibition of sialylation as well as chemical blocking of sialic acids reduced rapid cytoplasmic uptake, which could be reconstituted by thapsigargin. These results establish a link between the known propensity of arginine-rich CPPs to interact with the glycocalyx and calcium influx as the initial step triggering direct cytosolic peptide uptake., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented: (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy., (© 2020 Elsevier Inc. All rights reserved.)

Significance: In addition to their classical role in cellular ATP production, mitochondria are of key relevance in various (patho)physiological mechanisms including second messenger signaling, neuro-transduction, immune responses and death induction. Recent Advances: Within cells, mitochondria are motile and display temporal changes in internal and external structure ("mitochondrial dynamics"). During the last decade, substantial empirical and in silico evidence was presented demonstrating that mitochondrial dynamics impacts on mitochondrial function and vice versa . Critical Issues: However, a comprehensive and quantitative understanding of the bidirectional links between mitochondrial external shape, internal structure and function ("morphofunction") is still lacking. The latter particularly hampers our understanding of the functional properties and behavior of individual mitochondrial within single living cells. Future Directions: In this review we discuss the concept of mitochondrial morphofunction in mammalian cells, primarily using experimental evidence obtained within the last decade. The topic is introduced by briefly presenting the central role of mitochondria in cell physiology and the importance of the mitochondrial electron transport chain (ETC) therein. Next, we summarize in detail how mitochondrial (ultra)structure is controlled and discuss empirical evidence regarding the equivalence of mitochondrial (ultra)structure and function. Finally, we provide a brief summary of how mitochondrial morphofunction can be quantified at the level of single cells and mitochondria, how mitochondrial ultrastructure/volume impacts on mitochondrial bioreactions and intramitochondrial protein diffusion, and how mitochondrial morphofunction can be targeted by small molecules.

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: During its 120 days sojourn in the circulation, the red blood cell (RBC) remodels its membrane. Acetylcholinesterase (AChE) is a glycosylphosphatidylinositol (GPI)-linked enzyme that may serve as a marker for membrane processes occurring this ageing-associated remodelling process., Materials and Methods: Expression and enzymatic activity of AChE were determined on RBCs of various ages, as obtained by separation based on volume and density (ageing in vivo), and on RBCs of various times of storage in blood bank conditions (ageing in vitro), as well as on RBC-derived vesicles., Results: During ageing in vivo, the enzymatic activity of AChE decreases, but not the AChE protein concentration. In contrast, neither AChE activity nor concentration show a consistent, significant decrease during ageing in vitro. CD59, another GPI-linked protein that protects against complement-induced removal, also remains constant during storage. The cellular content of the integral membrane protein glycophorin A, however, decreases with storage time in the more dense RBC fractions. The latter are enriched in echinocytes and other misshapen cells during storage., Discussion: Our findings suggest that, during RBC ageing, GPI-linked proteins and integral membrane proteins are differentially sorted. Also, the vesicles that are generated in vitro show a fast and extensive loss of AChE activity, but not of AChE expression. Thus, AChE characteristics may constitute sensitive biomarkers of RBC ageing in vivo, and a source of information on the structural and functional changes that GPI-linked proteins undergo during ageing in vivo and in vitro. This information may help to understand RBC homeostasis and the effects of transfusion, especially in immunologically compromised patients.

To this point, efforts to develop therapeutic peptides for intracellular applications were guided by the perception that unmodified linear peptides are highly unstable and therefore structural modifications are required to reduce proteolytic breakdown. Largely, this concept is a consequence of the fact that most research on intracellular peptides hitherto has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Interestingly, inside cells, endogenous peptides lacking any chemical modifications to enhance stability escape degradation to the point that they may even modulate intracellular signaling pathways. In addition, many unmodified synthetic peptides designed to interfere with intracellular signaling, following introduction into cells, have the expected activity demonstrating that biologically relevant concentrations can be reached. This review provides an overview of results and techniques relating to the exploration and application of linear, unmodified peptides. After an introduction to intracellular peptide turnover, the review mentions examples for synthetic peptides as modulators of intracellular signaling, introduces endogenous peptides with bioactivity, techniques to measure peptide stability, and peptide delivery. Future experiments should elucidate the rules needed to predict promising peptide candidates., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding., (Copyright © 2014. Published by Elsevier B.V.)

Signals relayed through G protein-coupled receptors (GPCR) play pivotal roles in human physiology and are important drug targets. About 40% of all GPCRs couple to the heterotrimeric G protein Gq. Biochemical studies as well as crystallography have improved our understanding of GqPCRs and their downstream partners. Here we focus on the "functional imaging" tools that have been developed to visualize, dissect and quantify signalling processes at the single living cell level. We provide an overview of the most important developments in readout of signalling by FRET and BRET, as well as of the labelling strategies commonly used to visualize proteins in living cells. In addition, tools that allow manipulation of individual steps, including chemically inducible dimerization and optogenetic tools are covered. Together, these developments will provide unprecedented insights in GqPCR signalling in living cells and model organisms.

Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins will greatly overestimate connectivity within the network. Here, we addressed the impact of intracellular complexity on signalling networks using microarrays that carried a collection of peptides binding to the GRB2 SH2 and SH3 domains. Binding patterns and affinities for the recombinant adaptor protein GRB2 were compared with the ones for the protein in cell lysates. Peptide microarrays were titrated with the histidine-tagged recombinant protein, cell lysates or mixtures of both. Indeed, for recombinant GRB2, binding was detected for more peptides than for GRB2 in cell lysates. Moreover, binding was also observed for poor binders. It was impossible to define affinity thresholds for the binding of the recombinant protein to enable a discrimination of physiologically relevant interactions. Titrations of recombinant protein with lysate confirmed competition as the basis for fewer interactions. Importantly, the methods presented here enable the description of physiologically relevant binding patterns for proteins of interest and the identification of those peptide motifs, which are most strongly affected by competition., Biological Significance: The biological significance of protein-protein interactions can only be addressed in a physiologically meaningful way in the presence of the endogenous proteome which may contain proteins that compete for binding sites. Using peptide microarrays, we here demonstrate for the adaptor protein GRB2 that this competition strongly reduces the number of interactions with other signalling proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Phospholipase C (PLC) β isoforms are implicated in various physiological processes and pathologies. However, mechanistic insight into the localization and activation of each of the isoforms is limited. Therefore, it is crucial to gain more in-depth knowledge as to the regulation of the different isoforms. Here we describe the subcellular location of full-length PLCβ isozymes and their C-terminal (CT) domains. Strikingly, we found isoforms PLCβ1 and PLCβ4 to be enriched at the plasma membrane, contrary to isoforms PLCβ2 and PLCβ3. We determined that the CT domain is an inhibitor of Gq-mediated increases in intracellular calcium, the potency of its effect being dependent upon the CT domain isoform used. Furthermore, ratiometric fluorescence resonance energy transfer (FRET) imaging was used to study the kinetics of the Gαq-CTβx interactions. By the use of recently developed tools, which enable the on-demand activation of Gαq, we could show that the interaction between constitutively active Gαq and PLCβ3 prolongs the residence time of PLCβ3 at the plasma membrane. These findings suggest that under physiological circumstances, PLCβ3 and Gαq interact in a kiss-and-run fashion, likely due to the GTPase-activating activity of PLCβ towards Gαq., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The determination of intracellular protein concentrations is a prerequisite for understanding protein interaction networks in systems biology. Today, protein quantification is based either on mass spectrometry, which requires large cell numbers and sophisticated measurement protocols, or on quantitative Western blotting, which requires the expression and purification of a recombinant protein as a reference. Here, we present a method that uses a transiently expressed fluorescent fusion protein of the protein-of-interest as an easily accessible reference in small volumes of crude cell lysates. The concentration of the fusion protein is determined by fluorescence correlation spectroscopy, and this concentration is used to calibrate the intensity of bands on a Western blot. We applied this method to address cellular protein homeostasis by determining the concentrations of the plasma membrane-located transmembrane scaffolding protein LAT and soluble signaling proteins in naïve T cells and transformed T-cell lymphoma (Jurkat) cells (with the latter having nine times the volume of the former). Strikingly, the protein numbers of soluble proteins scaled with the cell volume, whereas that of the transmembrane protein LAT scaled with the membrane surface. This leads to significantly different stoichiometries of signaling proteins in transformed and naïve cells in concentration ranges that may translate directly into differences in complex formation., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)