RESUMO
Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/deficiência , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Animais , Biomarcadores , Caspases/metabolismo , Movimento Celular/genética , Movimento Celular/imunologia , Citocinas/genética , Citocinas/metabolismo , Suscetibilidade a Doenças , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Transdução de SinaisRESUMO
Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane. This article has an associated First Person interview with the first author of the paper.
Assuntos
Fibrose Cística , Aminofenóis/farmacologia , Benzodioxóis/farmacologia , Ceramidas , Análise por Conglomerados , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Lipídeos , Mutação/genéticaRESUMO
Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.
RESUMO
Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor's spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-ß-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR's ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-ß-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.
Assuntos
Colesterol , Receptores de LDL , Humanos , Colesterol/metabolismo , Clatrina/metabolismo , Fluorescência , Células HEK293 , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismoRESUMO
The mechanisms by which angiotensin II type 1 receptor is distributed and the diffusional pattern in the plasma membrane (PM) remain unclear, despite their crucial role in cardiovascular homeostasis. In this work, we obtained quantitative information of angiotensin II type 1 receptor (AT1R) lateral dynamics as well as changes in the diffusion properties after stimulation with ligands in living cells using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study the organization of the receptor at the nanoscale, expansion microscopy (ExM) combined with PALM was performed. This study revealed that AT1R lateral diffusion increased after binding to angiotensin II (Ang II) and the receptor diffusion was transiently confined in the PM. In addition, ExM revealed that AT1R formed nanoclusters at the PM and the cluster size significantly decreased after Ang II treatment. Taking these results together suggest that Ang II binding and activation cause reorganization and changes in the dynamics of AT1R at the PM.
Assuntos
Angiotensina II , Receptor Tipo 1 de Angiotensina , Receptor Tipo 1 de Angiotensina/metabolismo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Microscopia , Membrana Celular/metabolismoRESUMO
Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.
Assuntos
Cálcio/metabolismo , Imagem Óptica/métodos , Animais , Encéfalo/metabolismo , Caenorhabditis elegans/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Indicadores e Reagentes , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Optogenética , Engenharia de Proteínas , Espectroscopia de Luz Próxima ao Infravermelho , Xenopus laevis/metabolismoRESUMO
Dopamine (DA) is a key regulator of circuits controlling movement and motivation. A subset of midbrain DA neurons has been shown to express the vesicular glutamate transporter (VGLUT)2, underlying their capacity for glutamate release. Glutamate release is found mainly by DA neurons of the ventral tegmental area (VTA) and can be detected at terminals contacting ventral, but not dorsal, striatal neurons, suggesting the possibility that target-derived signals regulate the neurotransmitter phenotype of DA neurons. Whether glutamate can be released from the same terminals that release DA or from a special subset of axon terminals is unclear. Here, we provide in vitro and in vivo data supporting the hypothesis that DA and glutamate-releasing terminals in mice are mostly segregated and that striatal neurons regulate the cophenotype of midbrain DA neurons and the segregation of release sites. Our work unveils a fundamental feature of dual neurotransmission and plasticity of the DA system.-Fortin, G. M., Ducrot, C., Giguère, N., Kouwenhoven, W. M., Bourque, M.-J., Pacelli, C., Varaschin, R. K., Brill, M., Singh, S., Wiseman, P. W., Trudeau, L.-E. Segregation of dopamine and glutamate release sites in dopamine neuron axons: regulation by striatal target cells.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Ácido Glutâmico/metabolismo , Transmissão Sináptica , Área Tegmentar Ventral/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/fisiologia , Animais , Corpo Estriado/citologia , Neurônios Dopaminérgicos/citologia , Masculino , Camundongos , Camundongos Knockout , Área Tegmentar Ventral/citologiaRESUMO
The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.
Assuntos
Fenômenos Mecânicos , Purinas/metabolismo , Transdução de Sinais , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologiaRESUMO
Fluorescence fluctuation spectroscopy can be used to measure the aggregation of fluorescently labeled molecules and is typically performed using time series data. Spatial intensity distribution analysis and fluorescence moment image analysis are established tools for measuring molecular brightnesses from single-color images collected with laser scanning microscopes. We have extended these tools for analysis of two-color images to resolve heteromeric interactions between molecules labeled with spectrally distinct chromophores. We call these new methods two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA). To implement these techniques on a hyperspectral imaging system, we developed a spectral shift filtering technique to remove artifacts due to intrinsic cross talk between detector bins. We determined that 2c-SpCA provides better resolution from samples containing multiple fluorescent species; hence, this technique was carried forward to study images of living cells. We used fluorescent heterodimers labeled with enhanced green fluorescent protein and mApple to quantify the effects of resonance energy transfer and incomplete maturation of mApple on brightness measurements. We show that 2c-SpCA can detect the interaction between two components of trimeric G-protein complexes. Thus, 2c-SpCA presents a robust and computationally expedient means of measuring heteromeric interactions in cellular environments.
Assuntos
Algoritmos , Proteínas de Membrana/química , Multimerização Proteica , Membrana Celular/metabolismo , Cor , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , HumanosRESUMO
Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsa-stained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.
Assuntos
Citometria por Imagem/métodos , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Eritrócitos/química , Hemeproteínas/química , Hemoglobinas/química , Humanos , Processamento de Imagem Assistida por Computador , Estudo de Prova de Conceito , Esquizontes/isolamento & purificação , Software , Trofozoítos/isolamento & purificaçãoRESUMO
Since its invention over a hundred years ago, histological analysis using coloured dye staining remains the gold standard for histopathology. While these stains provide critical information for a variety of diagnostic purposes, they offer limited two-dimensional histological information. Extending classical histological analysis to three dimensions requires novel imaging approaches such as multiphoton microscopy. Multiphoton microscopy enables multimodal, three-dimensional imaging of histologically stained samples. Specifically, third harmonic generation (THG), a nonlinear optical process in which three incident photons are combined into one by the sample, allows high contrast imaging of tissues stained with absorbing dyes, which in turn act as harmonophores. While this technique has previously been applied to hematoxylin and eosin (H&E) tissue sections, we extend this approach to other commonly used histological stains to demonstrate further potential applications of the technique. We demonstrate THG imaging of both human skin and liver tissue stained with H&E, Verhoeff-Van Gieson (VVG) and Picrosirius Red stains. We find that these stains provide excellent contrast as THG harmonophores, enabling high resolution imaging of histological samples. THG imaging of the Verhoeff stain enables easy detection of elastic fibers while Picrosirius Red acts as an effective harmonophore for imaging collagen fibers of all sizes.
Assuntos
Corantes/química , Fígado/citologia , Pele/citologia , Colágeno/química , Tecido Elástico/citologia , Humanos , Fígado/química , Microscopia de Geração do Segundo Harmônico/métodos , Pele/química , Coloração e RotulagemRESUMO
Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/métodos , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Espectrometria de Fluorescência/métodos , Molécula 1 de Interação Estromal/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular , Simulação por Computador , Difusão , Células Epiteliais , Humanos , Microscopia Intravital/instrumentação , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Cell division in plant cells requires the deposition of a new cell wall between the two daughter cells. The assembly of this plate requires the coordinated movement of cargo vesicles whose size is below the diffraction-limited resolution of the optical microscope. We combined high spatial and temporal resolution confocal laser scanning microscopy with advanced image-processing tools and fluorescence fluctuation methods and distinguished three distinct phases during cell plate expansion in tobacco (Nicotiana tabacum) 'Bright Yellow-2' cells: massive delivery of preexisting vesicles to a disk-shaped region at the equatorial plane precedes a primary rapid expansion phase followed by a secondary, slow expansion phase during which the extremity of the circular plate seeks contact with the mother wall and brings about the separation of the two portions of cytoplasm. Different effects of pharmacological inhibition emphasize the distinct nature of the assembly and expansion mechanisms characterizing these phases.
Assuntos
Citocinese , Vesículas Citoplasmáticas/metabolismo , Células Vegetais/metabolismo , Desenvolvimento Vegetal , Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitose , Recuperação de Fluorescência Após Fotodegradação , Biossíntese de Proteínas , Análise Espectral , Fatores de Tempo , Nicotiana/citologia , Nicotiana/metabolismoRESUMO
The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T cells. Building on previous studies at the T cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component, α-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Sinapses Imunológicas/metabolismo , Linfócitos T/metabolismo , Actinina/genética , Actinina/metabolismo , Membrana Celular/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Células Jurkat , Microscopia de Fluorescência , Movimento (Física) , Imagem Individual de Molécula , Análise Espectral , Linfócitos T/efeitos dos fármacos , Moduladores de Tubulina/farmacologiaRESUMO
A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.
Assuntos
DNA/química , Corantes Fluorescentes/química , Nanotubos/química , Nanotecnologia/métodos , Nanotubos/ultraestrutura , Imagem Óptica , FotodegradaçãoRESUMO
Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.
Assuntos
Fatores de Crescimento Neural/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Membrana Celular/metabolismo , Receptor DCC , Células HEK293 , Humanos , Receptores de Netrina , Netrina-1 , Transporte ProteicoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.
Assuntos
Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Microdomínios da Membrana/metabolismo , Doença Aguda , Adenoviridae , Infecções por Adenovirus Humanos/metabolismo , Brônquios/metabolismo , Brônquios/virologia , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microdomínios da Membrana/virologia , Microscopia Confocal , Análise Espectral/métodosRESUMO
Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique-spatial intensity distribution analysis (SpIDA)-that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Proteína Proteolipídica de Mielina/química , Multimerização Proteica , Animais , Artefatos , Células COS , Chlorocebus aethiops , Simulação por Computador , Dimerização , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Microscopia Confocal/métodos , Modelos Biológicos , Mutação , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Fotodegradação , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Análise de Célula ÚnicaRESUMO
Whereas both GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) play a role in control of dorsal horn neuron excitability, their relative contribution to inhibition of small diameter primary afferent terminals remains controversial. To address this, we designed an approach for quantitative analyses of the distribution of GABA(A)R-subunits, GlyR α1-subunit and their anchoring protein, gephyrin, on terminals of rat spinal sensory afferents identified by Calcitonin-Gene-Related-Peptide (CGRP) for peptidergic terminals, and by Isolectin-B4 (IB4) for nonpeptidergic terminals. The approach was designed for light microscopy, which is compatible with the mild fixation conditions necessary for immunodetection of several of these antigens. An algorithm was designed to recognize structures with dimensions similar to those of the microscope resolution. To avoid detecting false colocalization, the latter was considered significant only if the degree of pixel overlap exceeded that expected from randomly overlapping pixels given a hypergeometric distribution. We found that both CGRP(+) and IB4(+) terminals were devoid of GlyR α1-subunit and gephyrin. The α1 GABA(A)R was also absent from these terminals. In contrast, the GABA(A)R α2/α3/α5 and ß3 subunits were significantly expressed in both terminal types, as were other GABA(A)R-associated-proteins (α-Dystroglycan/Neuroligin-2/Collybistin-2). Ultrastructural immunocytochemistry confirmed the presence of GABA(A)R ß3 subunits in small afferent terminals. Real-time quantitative PCR (qRT-PCR) confirmed the results of light microscopy immunochemical analysis. These results indicate that dorsal horn inhibitory synapses follow different rules of organization at presynaptic versus postsynaptic sites (nociceptive afferent terminals vs inhibitory synapses on dorsal horn neurons). The absence of gephyrin clusters from primary afferent terminals suggests a more diffuse mode of GABA(A)-mediated transmission at presynaptic than at postsynaptic sites.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Neurônios Aferentes/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA-A/metabolismo , Medula Espinal/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Lectinas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Glicina/metabolismoRESUMO
Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.