Nanoclustering as a dominant feature of plasma membrane organization

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.Postprint (published version

A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

PTP1B promotes focal complex maturation, lamellar persistence and directional migration

Previous findings established that ER-bound PTP1B targets peripheral cell-matrix adhesions and positively regulates cell adhesion to fibronectin. Here we show that PTP1B enhances focal complex lifetime at the lamellipodium base, delaying their turnover and facilitating α-actinin incorporation. We demonstrate the presence of catalytic PTP1BD181A-α-actinin complexes at focal complexes. Kymograph analysis revealed that PTP1B contributes to lamellar protrusion persistence and directional cell migration. Pull-down and FRET analysis also showed that PTP1B is required for efficient integrin-dependent downregulation of RhoA and upregulation of Rac1 during spreading. A substrate trap strategy revealed that FAK/Src recruitment and Src activity are essential for the generation of PTP1B substrates in adhesions. PTP1B targets the negative regulatory site of Src (phosphotyrosine 529), paxillin and p130Cas at peripheral cell-matrix adhesions. We postulate that PTP1B modulates more than one pathway required for focal complex maturation and membrane protrusion, including α-actinin-mediated cytoskeletal anchorage, integrin-dependent activation of the FAK/Src signaling pathway, and RhoA and Rac1 GTPase activity. By doing so, PTP1B contributes to coordinated adhesion turnover, lamellar stability and directional cell migration.Fil: Burdisso, Juan Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: González, Angela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Arregui, Carlos Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

The macroscopic effects of microscopic heterogeneity

Over the past decade, advances in super-resolution microscopy and particle-based modeling have driven an intense interest in investigating spatial heterogeneity at the level of single molecules in cells. Remarkably, it is becoming clear that spatiotemporal correlations between just a few molecules can have profound effects on the signaling behavior of the entire cell. While such correlations are often explicitly imposed by molecular structures such as rafts, clusters, or scaffolds, they also arise intrinsically, due strictly to the small numbers of molecules involved, the finite speed of diffusion, and the effects of macromolecular crowding. In this chapter we review examples of both explicitly imposed and intrinsic correlations, focusing on the mechanisms by which microscopic heterogeneity is amplified to macroscopic effect.Comment: 20 pages, 5 figures. To appear in Advances in Chemical Physic

Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

Indexación: Web of Science.Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.http://nanoscalereslett.springeropen.com/articles/10.1186/s11671-016-1260-

Heteroreceptor complexes and their allosteric receptor-receptor interactions in the central nervous system. Focus on examples from Dopamine D2 and Serotonin 5-HT1a receptors

GPCR interacting proteins (specially β- arrestin) and their receptor-protein interactions are also covered but their interactions with the allosteric receptor-receptor interactions in heteroreceptor complexes remain to be elucidated. The physiological and pathological relevance of the allosteric receptor-receptor interactions in heteroreceptor complexes is emphasized and novel strategies for treatment of mental and neurological disease are introduced based on this new biological principle of integration. This work gives further experimental evidences which strongly support the current view that allosteric receptor–receptor interactions in heteroreceptor complexes appear to represent a new principle in biology making possible integration of signals already at the level of the plasma membrane. These heteroreceptor complexes and their dynamics may be part of the molecular basis of learning and memory. The receptor protomers and their allosteric receptor-receptor interactions can be disturbed in neurological and mental disorders, and in diseases of peripheral tissues like the endocrine, cardiovascular and immune systems. The dopamine (DA) neuron system most relevant for schizophrenia and Parkinson s diseases is the meso-limbic-cortical DA system inter alia densely innervating subcortical limbic regions as well as the dorsal striatum. The field of dopamine D2Rs changed significantly with the discovery of many types of D2R heteroreceptor complexes in the ventral and dorsal striatum. The results indicate that the D2R is a hub receptor (www.gpcr-hetnet.com) which interacts not only with many other GPCRs including DA isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and DA transporters. Disturbances in several of these D2R heteroreceptor complexes may contribute to the development of schizophrenia and Parkinson s diseases through changes in the balance of diverse D2R homo- and heteroreceptor complexes mediating the DA signal, especially to the ventral striato-pallidal GABA pathway. In schizophrenia, this will have consequences for the control of this pathway of the glutamate drive to the prefrontal cortex via the mediodorsal thalamic nucleus which can contribute to psychotic processes. Allosteric receptor-receptor interactions in GPCR heteromers appeared to introduce an intermolecular allosteric mechanism contributing to the diversity and bias in the GPCR protomers. In A2A-D2R heteroreceptor complexes allosteric A2A-D2R receptor-receptor interaction brings about a biased modulation of the D2R protomer signalling (Chapter 1). A conformational state of the D2R is induced which moves away from Gi/o signaling and instead favours b-arrestin2 mediated signalling which may be the main mechanism for its atypical antipsychotic properties especially linked to the limbic A2AR-D2R heteroreceptor complexes. Furthermore, D2R-NTS1R heterocomplexes also exist in the ventral and dorsal striatum (Chapter 2) and likely also in midbrain DA nerve cells as D2R-NTS1R autoreceptor complexes where neurotensin produces antipsychotic and propsychotic actions, respectively. D2R protomer appeared to bias the specificity of the NT orthosteric binding site towards neuromedin N vs neurotensin in the heteroreceptor complex. There is a new awareness that Receptor tyrosine kinases (RTK) and transmitter activated GPCR possess the capacity for transactivation not only via GPCR induced release of neurotrophic factors, but also during signal initiation and propagation, using shared signaling pathways or using themselves as signaling platforms via direct allosteric receptor–receptor interactions. RTK are a family of transmembrane- spanning receptors that mediate the signaling from ligands such as growth factors, like the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), the brain derived neurotrophic factor (BDNF), and the fibroblast growth factor (FGF). This hypothesis on direct GPCR-RTK receptor-receptor interactions in heteroreceptor complexes was introduced by Fuxe et al 1983. They also proposed the existence of 5- HT1A-FGFR1 heteroreceptor complexes having a role in depression. The hypothesis was introduced that the neurotrophic system FGF-2/FGFR1 may be a good candidate to mediate antidepressant induced improvement in 5-HT neuronal communication and neurotrophism with regeneration of connections lost during depression. RTK transactivation in response to antidepressant drug treatment was postulated to take place via a new allosteric receptor–receptor between distinct serotonin receptor subtypes and FGFR1 in heteroreceptor complexes. The discovery of brain FGFR1-5-HT1A heteroreceptor complexes and their enhancement of neuroplasticity offers an integration of the serotonin and the neurotrophic factor hypotheses of depression at the molecular level. These heteroreceptor complexes were found in the hippocampus and midbrain raphe 5-HT nerve cells, enriched in 5-HT1A autoreceptors. Based on the triplet puzzle theory several sets of triplet homologies were identified that may be part of the receptor interface. Combined FGF-2 and 5-HT1A agonist treatment increased the formation of these heterocomplexes and the facilitatory allosteric receptor-receptor interactions within them leading to an enhancement of FGFR1 signaling (Chapter 3). This integrative phenomenon is reciprocal and RTK signaling can be placed downstream of GPCRs. Formation of such heterocomplexes involving two major classes of membrane receptors can be involved in regulating all aspects of receptor protomer function including recognition, signaling, trafficking, desensitization, and downregulation (Chapter 3). These events were associated with development of rapid antidepressant effects. These heteroreceptor complexes are a novel target for antidepressant drugs. These examples, based on solid experimental evidences, serve to illustrate that allosteric receptor-receptor interactions in GPCR heteroreceptor complexes play a significant role in receptor diversity and bias of the participating GPCR protomers.G-protein coupled receptors (GPCR)-mediated signalling is a more complicated process than described previously since every GPCR and GPCR heteromer requires a set of G protein interacting proteins (GIP) which interacts with the receptor in an orchestrated spatio-temporal fashion. Therefore, there is a high interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time, and specially, their integration in GPCR heterocomplexes of the Central Nervous System (CNS). Also, pathological protein-protein interactions in homocomplexes and heterocomplexes of Aβ, Tau, and α-Syn are at the heart of the development of conformational protein disorders. Along this work, experimental evidences are given to illustrate that GPCR interactions have relevance for neurological and mental diseases and are targets for drug development. GPCR containing heteromers and higher order heteromers through allosteric receptor- receptor interactions have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. They have become exciting new targets for neurotherapeutics in e.g. Parkinson’s disease, schizophrenia, drug addiction, anxiety and depression opening up a new field in neuropsychopharmacology. Along this work, the allosteric receptor-receptor interactions over the interfaces in A2AR-D2R, D2R-NTS1R, D2R-Sigma1R and 5-HT1A-FGFR1 heteroreceptor complexes will be explored and their biochemical, pharmacological and functional integrative implications in the CNS described. Methodologies for studies on receptor- receptor interactions are discussed including the use of FRET and BRET-based techniques in the analysis of G protein coupled receptor (GPCR) dimerization in living cells. In situ proximity ligation assay is performed to establish the existence of native heteroreceptor complexes in the CNS

Inferring diffusion in single live cells at the single molecule level

The movement of molecules inside living cells is a fundamental feature of biological processes. The ability to both observe and analyse the details of molecular diffusion in vivo at the single molecule and single cell level can add significant insight into understanding molecular architectures of diffusing molecules and the nanoscale environment in which the molecules diffuse. The tool of choice for monitoring dynamic molecular localization in live cells is fluorescence microscopy, especially so combining total internal reflection fluorescence (TIRF) with the use of fluorescent protein (FP) reporters in offering exceptional imaging contrast for dynamic processes in the cell membrane under relatively physiological conditions compared to competing single molecule techniques. There exist several different complex modes of diffusion, and discriminating these from each other is challenging at the molecular level due to underlying stochastic behaviour. Analysis is traditionally performed using mean square displacements of tracked particles, however, this generally requires more data points than is typical for single FP tracks due to photophysical instability. Presented here is a novel approach allowing robust Bayesian ranking of diffusion processes (BARD) to discriminate multiple complex modes probabilistically. It is a computational approach which biologists can use to understand single molecule features in live cells.Comment: combined ms (1-37 pages, 8 figures) and SI (38-55, 3 figures

Translational Oncogenomics and Human Cancer Interactome Networks

An overview of translational, human oncogenomics, transcriptomics and cancer interactomic networks is presented together with basic concepts and potential, new applications to Oncology and Integrative Cancer Biology. Novel translational oncogenomics research is rapidly expanding through the application of advanced technology, research findings and computational tools/models to both pharmaceutical and clinical problems. A self-contained presentation is adopted that covers both fundamental concepts and the most recent biomedical, as well as clinical, applications. Sample analyses in recent clinical studies have shown that gene expression data can be employed to distinguish between tumor types as well as to predict outcomes. Potentially important applications of such results are individualized human cancer therapies or, in general, ‘personalized medicine’. Several cancer detection techniques are currently under development both in the direction of improved detection sensitivity and increased time resolution of cellular events, with the limits of single molecule detection and picosecond time resolution already reached. The urgency for the complete mapping of a human cancer interactome with the help of such novel, high-efficiency / low-cost and ultra-sensitive techniques is also pointed out

Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Acknowledgements We thank Marco Thiel for assistance with data interpretation, Peter Sudbery for the provision of strains and Jeremy Craven for useful discussions. This work was supported by a BBSRC-DTG to D. D. T., NIH award DK083592 to F. J. B. and P. A. J., and a Royal Society URF UF080611 and MRC NIRG 90671 to A. C. B.Non peer reviewedPublisher PD