RESUMO
Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.
Assuntos
Proteínas de Drosophila , Proteínas de Homeodomínio , Animais , Padronização Corporal/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Imaging fluorescence correlation spectroscopy (FCS) is a powerful tool to extract information on molecular mobilities, actions, and interactions in live cells, tissues, and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1-ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Third, as FCS evaluation is model dependent, data evaluation is significantly slowed unless analytic models are available. Here, we introduce two convolutional neural networks-FCSNet and ImFCSNet-for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both convolutional neural networks are trained on simulated data, are model agnostic, and allow autonomous, real-time evaluation of imaging FCS measurements.
Assuntos
Aprendizado Profundo , Espectrometria de Fluorescência/métodosRESUMO
Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control.
RESUMO
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.
Assuntos
Vírus da Dengue , Dengue , Flavivirus , Animais , Humanos , Vírus da Dengue/metabolismo , Proteínas do Envelope Viral/metabolismo , Gangliosídeos/metabolismo , Flavivirus/metabolismo , Mamíferos/metabolismoRESUMO
Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.
Assuntos
Ansiedade/patologia , Orientação de Axônios , Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/patologia , Proteínas do Tecido Nervoso/deficiência , Neurogênese , Proteínas de Peixe-Zebra/deficiência , Animais , Ansiedade/etiologia , Ansiedade/metabolismo , Sistemas CRISPR-Cas , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.
Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Chaperonas Moleculares/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Recombinação Genética/genética , Replicação Viral/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Códon de Iniciação , Ciclização/genética , Vírus da Dengue/genética , Cinética , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Modern electron-multiplying charge-coupled device (EMCCD) and scientific complementary metal-oxide semiconductor (sCMOS) cameras read out fluorescence data with single-molecule sensitivity at thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for total internal reflection fluorescence microscopy (TIRFM) and single-plane illumination microscopy (SPIM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses imaging fluorescence correlation spectroscopy for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRFM and SPIM data acquisition.
Assuntos
Elétrons , Semicondutores , Desenho de Equipamento , Microscopia de Fluorescência/métodos , Espectrometria de FluorescênciaRESUMO
Number and brightness (N&B) analysis is a fluorescence spectroscopy technique to quantify oligomerization of the mobile fraction of proteins. Accurate results, however, rely on a good knowledge of nonfluorescent states of the fluorescent labels, especially of fluorescent proteins, which are widely used in biology. Fluorescent proteins have been characterized for confocal, but not camera-based, N&B, which allows, in principle, faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment are still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor's oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization is not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, lead to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization dependent, involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation are the outcomes of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.
Assuntos
Receptores ErbBRESUMO
The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer-induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10-15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.
Assuntos
Colesterol , Microdomínios da Membrana , Membrana Celular/química , Colesterol/metabolismo , Heptanol/análise , Heptanol/metabolismo , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Membrana/metabolismo , Príons/metabolismo , Actinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Análise por Conglomerados , Citoesqueleto/metabolismo , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Humanos , Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Isoformas de Proteínas/metabolismo , Scrapie/metabolismoRESUMO
The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.
Assuntos
Regiões 5' não Traduzidas/genética , Proteínas do Capsídeo/fisiologia , Vírus da Dengue/metabolismo , Pareamento de Bases/genética , Sequência de Bases , Proteínas do Capsídeo/metabolismo , Sequência Conservada , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Genoma Viral/fisiologia , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA Circular/química , RNA Circular/genética , RNA Viral/química , RNA Viral/genética , Replicação Viral/genéticaRESUMO
Diffusion obstacles in membranes have not been directly visualized because of fast membrane dynamics and the occurrence of subresolution molecular complexes. To understand the obstacle characteristics, mobility-based methods are often used as an indirect way of assessing the membrane structure. Molecular movement in biological plasma membranes is often characterized by anomalous diffusion, but the exact underlying mechanisms are still elusive. Imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is a well-established mobility-based method that provides spatially resolved diffusion coefficient maps and is combined with FCS diffusion law analysis to examine subresolution membrane organization. In recent years, although FCS diffusion law analysis has been instrumental in providing new insights into the membrane structure below the optical diffraction limit, there are certain exceptions and anomalies that require further clarification. To this end, we correlate the membrane structural features imaged by atomic force microscopy (AFM) with the dynamics measured using ITIR-FCS. We perform ITIR-FCS measurements on supported lipid bilayers (SLBs) of various lipid compositions to characterize the anomalous diffusion of lipid molecules in distinct obstacle configurations, along with the high-resolution imaging of the membrane structures with AFM. Furthermore, we validate our experimental results by performing simulations on image grids with experimentally determined obstacle configurations. This study demonstrates that FCS diffusion law analysis is a powerful tool to determine membrane heterogeneities implied from dynamics measurements. Our results corroborate the commonly accepted interpretations of imaging FCS diffusion law analysis, and we show that exceptions happen when domains reach the percolation threshold in a biphasic membrane and a network of domains behaves rather like a meshwork, resulting in hop diffusion.
Assuntos
Bicamadas Lipídicas , Membrana Celular , Difusão , Microscopia de Força Atômica , Espectrometria de FluorescênciaRESUMO
A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.
Assuntos
Membrana Celular/metabolismo , Lipídeos/química , Microscopia de Fluorescência , Animais , Linhagem Celular , Membrana Celular/química , Estrutura Molecular , Espectrometria de Fluorescência , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
G-Protein-coupled receptors (GPCRs) are ubiquitous within eukaryotes, responsible for a wide array of physiological and pathological processes. Indeed, the fact that they are the most drugged target in the human genome is indicative of their importance. Despite the clear interest in GPCRs, most information regarding their activity has been so far obtained by analyzing the response from a "bulk medium". As such, this Perspective summarizes some of the common methods for this indirect observation. Nonetheless, by inspecting approaches applying super-resolution imaging, we argue that imaging is perfectly situated to obtain more detailed structural and spatial information, assisting in the development of new GPCR-targeted drugs and clinical strategies. The benefits of direct optical visualization of GPCRs are analyzed in the context of potential future directions in the field.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Descoberta de Drogas/métodos , Humanos , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/ultraestrutura , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
Dehydrins are intrinsically disordered proteins, generally expressed in plants as a response to embryogenesis and water-related stress. Their suggested functions are in membrane stabilization and cell protection. All dehydrins contain at least one copy of the highly conserved K-segment, proposed to be a membrane-binding motif. The dehydrin Lti30 (Arabidopsis thaliana) is up-regulated during cold and drought stress conditions and comprises six K-segments, each with two adjacent histidines. Lti30 interacts with the membrane electrostatically via pH-dependent protonation of the histidines. In this work, we seek a molecular understanding of the membrane interaction mechanism of Lti30 by determining the diffusion and molecular organization of Lti30 on model membrane systems by imaging total internal reflection- fluorescence correlation spectroscopy (ITIR-FCS) and molecular dynamics (MD) simulations. The dependence of the diffusion coefficient explored by ITIR-FCS together with MD simulations yields insights into Lti30 binding, domain partitioning, and aggregation. The effect of Lti30 on membrane lipid diffusion was studied on fluorescently labeled supported lipid bilayers of different lipid compositions at mechanistically important pH conditions. In parallel, we compared the mode of diffusion for short individual K-segment peptides. The results indicate that Lti30 binds the lipid bilayer via electrostatics, which restricts the mobility of lipids and bound protein molecules. At low pH, Lti30 binding induced lipid microdomain formation as well as protein aggregation, which could be correlated with one another. Moreover, at physiological pH, Lti30 forms nanoscale aggregates when proximal to the membrane suggesting that Lti30 may protect the cell by "cross-linking" the membrane lipids.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Lipídeos de Membrana , Simulação de Dinâmica Molecular , Pressão Osmótica , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Lipídeos de Membrana/metabolismo , Domínios ProteicosRESUMO
How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly α coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.
Assuntos
Núcleo Celular/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Difusão , Humanos , Modelos Moleculares , Ligação Proteica , RNA Longo não Codificante/genética , Espectrometria de FluorescênciaRESUMO
The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τd) versus observation area. For at least three different diffusive modes - free, domain confined, and meshwork hindered hop diffusion - the expected plots have been characterized, typically by its y-intercept (τ0) when fit with a linear model, and have been verified in many cases. However, a description of τ0 has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ0 value is problematic. Here, we therefore address the question about the absolute value of τ0 in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ0 changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ0 is not influenced equally by the different diffusive modes as typically found in cellular environments, and that it is the relative change of τ0 rather than its absolute value that provides information on the mode of diffusion.
Assuntos
Membrana Celular/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Células CHO , Cricetulus , Citoesqueleto/metabolismo , Difusão , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Domínios Proteicos , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper's redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = - 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms. Graphical abstract.
Assuntos
Compostos de Boro/química , Cobre/análise , Corantes Fluorescentes/química , Oligopeptídeos/química , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Cátions Bivalentes/análise , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Oligopeptídeos/síntese químicaRESUMO
Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.
Assuntos
Blastocisto/metabolismo , Fatores de Transcrição SOXB1/genética , Animais , Células CHO , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Cricetinae , Cricetulus , Embrião de Mamíferos , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Gravidez , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genéticaRESUMO
The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.