RESUMO
Mouse pericentromeric DNA is composed of tandem major satellite repeats, which are heterochromatinized and cluster together to form chromocenters. These clusters are refractory to DNA repair through homologous recombination (HR). The mechanisms by which pericentromeric heterochromatin imposes a barrier on HR and the implications of repeat clustering are unknown. Here, we compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters. We show that while DSBs increase the accessibility of human pericentromeric heterochromatin by disrupting HP1α dimerization, mouse pericentromeric heterochromatin repeat clustering imposes a physical barrier that requires many layers of de-compaction to be accessed. Our results support a model in which the 3D organization of heterochromatin dictates the spatial activation of DNA repair pathways and is key to preventing the activation of HR within clustered repeats and the onset of chromosomal translocations.
Assuntos
Heterocromatina , Translocação Genética , Animais , Análise por Conglomerados , Quebras de DNA de Cadeia Dupla , Heterocromatina/genética , Recombinação Homóloga/genética , CamundongosRESUMO
Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.
Assuntos
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Nucleossomos , Transferência Ressonante de Energia de Fluorescência , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Epigênese GenéticaRESUMO
The thermal tolerance of symbiodiniacean photo-endosymbionts largely underpins the thermal bleaching resilience of their cnidarian hosts such as corals and the coral model Exaiptasia diaphana. While variation in thermal tolerance between species is well documented, variation between conspecific strains is understudied. We compared the thermal tolerance of three closely related strains of Breviolum minutum represented by two internal transcribed spacer region 2 profiles (one strain B1-B1o-B1g-B1p and the other two strains B1-B1a-B1b-B1g) and differences in photochemical and non-photochemical quenching, de-epoxidation state of photopigments, and accumulation of reactive oxygen species under rapid short-term cumulative temperature stress (26-40 °C). We found that B. minutum strains employ distinct photoprotective strategies, resulting in different upper thermal tolerances. We provide evidence for previously unknown interdependencies between thermal tolerance traits and photoprotective mechanisms that include a delicate balancing of excitation energy and its dissipation through fast relaxing and state transition components of non-photochemical quenching. The more thermally tolerant B. minutum strain (B1-B1o-B1g-B1p) exhibited an enhanced de-epoxidation that is strongly linked to the thylakoid membrane melting point and possibly membrane rigidification minimizing oxidative damage. This study provides an in-depth understanding of photoprotective mechanisms underpinning thermal tolerance in closely related strains of B. minutum.
Assuntos
Fotossíntese , Dinoflagellida/fisiologia , Resposta ao Choque Térmico , Temperatura AltaRESUMO
The mitochondrial inner membrane is a protein-rich environment containing large multimeric complexes, including complexes of the mitochondrial electron transport chain, mitochondrial translocases and quality control machineries. Although the inner membrane is highly proteinaceous, with 40-60% of all mitochondrial proteins localised to this compartment, little is known about the spatial distribution and organisation of complexes in this environment. We set out to survey the arrangement of inner membrane complexes using stochastic optical reconstruction microscopy (STORM). We reveal that subunits of the TIM23 complex, TIM23 and TIM44 (also known as TIMM23 and TIMM44, respectively), and the complex IV subunit COXIV, form organised clusters and show properties distinct from the outer membrane protein TOM20 (also known as TOMM20). Density based cluster analysis indicated a bimodal distribution of TIM44 that is distinct from TIM23, suggesting distinct TIM23 subcomplexes. COXIV is arranged in larger clusters that are disrupted upon disruption of complex IV assembly. Thus, STORM super-resolution microscopy is a powerful tool for examining the nanoscale distribution of mitochondrial inner membrane complexes, providing a 'visual' approach for obtaining pivotal information on how mitochondrial complexes exist in a cellular context.
Assuntos
Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial , Animais , Células HEK293 , Células HeLa , Humanos , Microscopia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transporte ProteicoRESUMO
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.
Assuntos
Glomerulonefrite/genética , Hipotricose/genética , Linfedema/genética , Fatores de Transcrição SOXF/genética , Telangiectasia/genética , Transcrição Gênica , Animais , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipotricose/metabolismo , Hipotricose/patologia , Luciferases/genética , Luciferases/metabolismo , Linfedema/metabolismo , Linfedema/patologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Mutação , Fatores de Transcrição SOXF/metabolismo , Imagem Individual de Molécula , Telangiectasia/metabolismo , Telangiectasia/patologiaRESUMO
Nuclear proteins can modulate their DNA binding activity and the exploration volume available during DNA target search by self-associating into higher-order oligomers. Directly tracking this process in the nucleoplasm of a living cell is, however, a complex task. Thus, here we present a microscopy method based on radial pair correlation of molecular brightness fluctuations (radial pCOMB) that can extract the mobility of a fluorescently tagged nuclear protein as a function of its oligomeric state and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture. By simply performing a rapid frame scan acquisition, radial pCOMB has the capacity to detect, within each pixel, protein oligomer formation and the size-dependent obstruction nuclear architecture imparts on this complex's transport across sub-micrometer distances. From application of radial pCOMB to an oligomeric transcription factor and DNA repair protein, we demonstrate that homo-oligomer formation differentially regulates chromatin accessibility and interaction with the DNA template.
Assuntos
Núcleo Celular , Proteínas Nucleares , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Difusão , Proteínas Nucleares/metabolismoRESUMO
To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.
Assuntos
Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Perilipin 5 (PLIN5) is a lipid-droplet-associated protein that coordinates intracellular lipolysis in highly oxidative tissues and is thought to regulate lipid metabolism in response to phosphorylation by protein kinase A (PKA). We sought to identify PKA phosphorylation sites in PLIN5 and assess their functional relevance in cultured cells and the livers of mice. We detected phosphorylation on S155 and identified S155 as a functionally important site for lipid metabolism. Expression of phosphorylation-defective PLIN5 S155A in Plin5 null cells resulted in decreased rates of lipolysis and triglyceride-derived fatty acid oxidation. FLIM-FRET analysis of protein-protein interactions showed that PLIN5 S155 phosphorylation regulates PLIN5 interaction with adipose triglyceride lipase at the lipid droplet, but not with α-ß hydrolase domain-containing 5. Re-expression of PLIN5 S155A in the liver of Plin5 liver-specific null mice reduced lipolysis compared with wild-type PLIN5 re-expression, but was not associated with other changes in hepatic lipid metabolism. Furthermore, glycemic control was impaired in mice with expression of PLIN5 S155A compared with mice expressing PLIN5. Together, these studies demonstrate that PLIN5 S155 is required for PKA-mediated lipolysis and builds on the body of evidence demonstrating a critical role for PLIN5 in coordinating lipid and glucose metabolism.
Assuntos
Perilipina-5RESUMO
Environmental polarity is an important factor that drives biomolecular interactions to regulate cell function. Herein, a general method of using the fluorogenic probe NTPAN-MI is reported to quantify the subcellular polarity change in response to protein unfolding. NTPAN-MI fluorescence is selectively activated upon labeling unfolded proteins with exposed thiols, thereby reporting on the extent of proteostasis. NTPAN-MI also reveals the collapse of the host proteome caused by influenza A virus infection. The emission profile of NTPAN-MI contains information of the local polarity of the unfolded proteome, which can be resolved through spectral phasor analysis. Under stress conditions that disrupt different checkpoints of protein quality control, distinct patterns of dielectric constant distribution in the cytoplasm can be observed. However, in the nucleus, the unfolded proteome was found to experience a more hydrophilic environment across all the stress conditions, indicating the central role of nucleus in the stress response process.
Assuntos
Proteoma , Resposta a Proteínas não Dobradas/genética , Núcleo Celular , Citoplasma , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Células HEK293 , HumanosRESUMO
Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.
Assuntos
Microscopia/métodos , Proteínas Nucleares/metabolismo , Espectrometria de Fluorescência/métodos , Fenômenos Biofísicos , Núcleo Celular/metabolismo , DNA/química , DNA/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição/genéticaRESUMO
Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross-pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Camundongos , Células NIH 3T3 , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Preparation of Nanographene oxide (NGO) - Gelatin hybrids for efficient treatment of Neuroblastoma. METHODS: Nanohybrids were prepared via non-covalent interactions. Spectroscopic tools have been used to discriminate the chemical states of NGO prior and after gelatin coating, with UV visible spectroscopy revealing the maximum binding capacity of gelatin to NGO. Raman and X-ray photoelectron spectroscopy (XPS) demonstrated NGO and Gelatin_NGO nanohybrids through a new chemical environments produced after noncovalent interaction. Microscopic analyses, atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to estimate the thickness of samples and the lateral width in the nanoscale, respectively. RESULTS: The cell viability assay validated Gelatin_NGO nanohybrids as a useful nanocarrier for Carboplatin (CP) release and delivery, without obvious signs of toxicity. The nano-sized NGO (200 nm and 300 nm) did not enable CP to kill the cancer cells efficiently, whilst the CP loaded Gel_NGO 100 nm resulted in a synergistic activity through increasing the local concentration of CP inside the cancer cells. CONCLUSIONS: The nanohybrids provoked high stability and dispersibility in physiological media, as well as enhanced the anticancer activity of the chemotherapy agent Carboplatin (CP) in human neuroblastoma cells.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Portadores de Fármacos , Gelatina/química , Grafite/química , Nanopartículas , Neuroblastoma/tratamento farmacológico , Óxidos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/metabolismo , Carboplatina/administração & dosagem , Carboplatina/química , Carboplatina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Relação Dose-Resposta a Droga , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Nanomedicina , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Espectroscopia Fotoeletrônica , Espectrofotometria Ultravioleta , Análise Espectral Raman , Tecnologia Farmacêutica/métodosRESUMO
The structure of cell membranes has been intensively investigated and many models and concepts have been proposed for the lateral organization of the plasma membrane. While proteomics and lipidomics have identified many if not all membrane components, how lipids and proteins interactions are coordinated in a specific cell function remains poorly understood. It is generally accepted that the organization of the plasma membrane is likely to play a critical role in the regulation of cell function such as receptor signalling by governing molecular interactions and dynamics. In this review we present different plasma membrane models and discuss microscopy approaches used for investigating protein behaviour, distribution and lipid organization.
Assuntos
Membrana Celular/ultraestrutura , Microdomínios da Membrana/ultraestrutura , Microscopia/métodos , Membrana Celular/química , Membrana Celular/genética , Metabolismo Energético , Humanos , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Modelos Biológicos , Mapas de Interação de Proteínas/genética , ProteômicaRESUMO
Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.
Assuntos
Núcleo Celular/genética , Cromatina/genética , Reparo de DNA por Recombinação , Cromatina/química , Quebras de DNA de Cadeia Dupla , Células HeLa , HumanosRESUMO
The Hippo pathway is an important regulator of organ growth and cell fate. The major mechanism by which Hippo is known to control transcription is by dictating the nucleo-cytoplasmic shuttling rate of Yorkie, a transcription co-activator, which promotes transcription with the DNA binding protein Scalloped. The nuclear biophysical behavior of Yorkie and Scalloped, and whether this is regulated by the Hippo pathway, remains unexplored. Using multiple live-imaging modalities on Drosophila tissues, we found that Scalloped interacts with DNA on a broad range of timescales, and enrichment of Scalloped at sites of active transcription is mediated by longer DNA dwell times. Further, Yorkie increased Scalloped's DNA dwell time, whereas the repressors Nervous fingers 1 (Nerfin-1) and Tondu-domain-containing growth inhibitor (Tgi) decreased it. Therefore, the Hippo pathway influences transcription not only by controlling nuclear abundance of Yorkie but also by modifying the DNA binding kinetics of the transcription factor Scalloped.
Assuntos
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Transativadores , Transcrição Gênica , Proteínas de Sinalização YAP , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas de Sinalização YAP/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA/metabolismo , DNA/genéticaRESUMO
Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.
RESUMO
In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.
Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Técnicas Biossensoriais/métodosRESUMO
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
RESUMO
Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan's emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan's emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan's fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.
Assuntos
2-Naftilamina/análogos & derivados , Membrana Celular/metabolismo , Colesterol/metabolismo , Corantes Fluorescentes/química , Lauratos/química , Fluidez de Membrana , 2-Naftilamina/química , Animais , Técnicas Biossensoriais , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Fator de Crescimento Epidérmico/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Fosfatidilcolinas/metabolismo , Soluções , Espectrometria de FluorescênciaRESUMO
No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.