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1.
Cell ; 177(4): 806-819, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-31051105

RESUMEN

Over the last several decades, an impressive array of advanced microscopic and analytical tools, such as single-particle tracking and nanoscopic fluorescence correlation spectroscopy, has been applied to characterize the lateral organization and mobility of components in the plasma membrane. Such analysis can tell researchers about the local dynamic composition and structure of membranes and is important for predicting the outcome of membrane-based reactions. However, owing to the unresolved complexity of the membrane and the structures peripheral to it, identification of the detailed molecular origin of the interactions that regulate the organization and mobility of the membrane has not proceeded quickly. This Perspective presents an overview of how cell-surface structure may give rise to the types of lateral mobility that are observed and some potentially fruitful future directions to elucidate the architecture of these structures in more molecular detail.


Asunto(s)
Membrana Celular/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/fisiología , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Proteínas de la Membrana/fisiología
2.
Nat Immunol ; 20(3): 350-361, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30718914

RESUMEN

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.


Asunto(s)
Agammaglobulinemia/inmunología , Linfocitos B/inmunología , Proteínas de Transporte de Catión/inmunología , Zinc/inmunología , Agammaglobulinemia/genética , Agammaglobulinemia/metabolismo , Animales , Linfocitos B/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Preescolar , Citosol/inmunología , Citosol/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Lactante , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Linaje , Zinc/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(28): 14002-14010, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31221762

RESUMEN

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Receptores de Antígenos de Linfocitos T/química , Animales , Humanos , Cinética , Ligandos , Activación de Linfocitos/genética , Complejo Mayor de Histocompatibilidad/inmunología , Microvellosidades/genética , Microvellosidades/inmunología , Modelos Teóricos , Péptidos/química , Péptidos/inmunología , Fosforilación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Imagen Individual de Molécula , Linfocitos T/química , Linfocitos T/inmunología
4.
J Phys D Appl Phys ; 50(6): 063001, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28458397

RESUMEN

Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, ⩾5 µm2 s-1, in the plasma membrane of live cells at very short length scales, ≈⩽ 100 nm, and time scales, ≈1-10 ms. This fast diffusion coefficient has been advocated in several high-speed SPT studies, for lipids and membrane proteins alike, but the equivalent has not been detected in STED-FCS measurements. Resolving this ambiguity is important because the assessment of membrane dynamics currently relies heavily on SPT for the determination of heterogeneous diffusion. A possible systematic error in this approach would thus have vast implications in this field. To address this, we have re-visited the analysis procedure for SPT data with an emphasis on the measurement errors and the effect that these errors have on the measurement outputs. We subsequently demonstrate that STED-FCS and SPT data, following careful consideration of the experimental errors of the SPT data, converge to a common interpretation which for the case of a diffusing phospholipid analogue in the plasma membrane of live mouse embryo fibroblasts results in an unhindered, intra-compartment, diffusion coefficient of ≈0.7-1.0 µm2 s-1, and a compartment size of about 100-150 nm.

5.
Methods ; 88: 67-75, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26123184

RESUMEN

Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.


Asunto(s)
Membrana Dobles de Lípidos/química , Microscopía Fluorescente/métodos , Espectrometría de Fluorescencia/métodos , Difusión
6.
J Immunol ; 191(8): 4165-73, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24043904

RESUMEN

Recent studies have shown that Abs that target the cell-surface enzyme CD73 (ecto-5'-nucleotidase) reduce growth of primary tumors and metastasis in syngenic mice by inhibiting the catalytic activity of CD73, and thus increasing the activity of cytotoxic T lymphocytes. In this article, we report another anticancer mechanism of anti-CD73 Abs and show that an anti-CD73 mAb (AD2) inhibits metastasis formation by a mechanism independent of CD73 catalytic activity and inhibition of primary tumor growth. This mechanism involves clustering and internalization of CD73, but does not require cross-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab' fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73 internalization and metastasis inhibition. Furthermore, anti-CD73 AD2 mAb inhibited development of metastasis in a spontaneous animal model of human metastatic breast cancer. Our study shows that some anti-CD73 mAbs cause cell-surface clustering of CD73 followed by internalization, thus inhibiting the ability of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.


Asunto(s)
5'-Nucleotidasa , Anticuerpos Monoclonales/inmunología , Neoplasias de la Mama/terapia , Metástasis de la Neoplasia/prevención & control , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/inmunología , 5'-Nucleotidasa/metabolismo , Animales , Transporte Biológico , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Movimiento Celular , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Metástasis de la Neoplasia/inmunología , Trasplante de Neoplasias , Células Neoplásicas Circulantes , Interferencia de ARN , ARN Interferente Pequeño , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Nano Lett ; 13(6): 2332-7, 2013 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-23647479

RESUMEN

In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several thousand steps) to allow for robust single trajectory analysis. This analysis indicates that a majority of the investigated molecules are transiently confined in nanoscopic compartments with a mean size of (100­150 nm)(2) for a mean duration of 50­100 ms.


Asunto(s)
Compartimento Celular , Membrana Celular/metabolismo , Puntos Cuánticos
8.
Nat Commun ; 15(1): 5392, 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38918391

RESUMEN

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.


Asunto(s)
Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN , Reparación del ADN , Exodesoxirribonucleasas , Humanos , Roturas del ADN de Doble Cadena/efectos de la radiación , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , ADN/metabolismo , ADN/genética , Ubiquitinación , Proteínas de Ciclo Celular
9.
Biochem Biophys Res Commun ; 430(3): 993-8, 2013 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-23261438

RESUMEN

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.


Asunto(s)
Acuaporinas/metabolismo , Células Epiteliales/fisiología , Agua/metabolismo , Cicatrización de Heridas , Animales , Movimiento Celular , Perros , Células Epiteliales/metabolismo , Células de Riñón Canino Madin Darby
10.
JCI Insight ; 8(9)2023 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-36976644

RESUMEN

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.


Asunto(s)
Células T Asesinas Naturales , Humanos , Células Presentadoras de Antígenos , Lipoproteínas/metabolismo
11.
J Cell Biol ; 221(9)2022 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-35861803

RESUMEN

Centrioles duplicate once per cell cycle, but it is unclear how daughter centrioles assemble at the right time and place and grow to the right size. Here, we show that in Drosophila embryos the cytoplasmic concentrations of the key centriole assembly proteins Asl, Plk4, Ana2, Sas-6, and Sas-4 are low, but remain constant throughout the assembly process-indicating that none of them are limiting for centriole assembly. The cytoplasmic diffusion rate of Ana2/STIL, however, increased significantly toward the end of S-phase as Cdk/Cyclin activity in the embryo increased. A mutant form of Ana2 that cannot be phosphorylated by Cdk/Cyclins did not exhibit this diffusion change and allowed daughter centrioles to grow for an extended period. Thus, the Cdk/Cyclin-dependent phosphorylation of Ana2 seems to reduce the efficiency of daughter centriole assembly toward the end of S-phase. This helps to ensure that daughter centrioles stop growing at the correct time, and presumably also helps to explain why centrioles cannot duplicate during mitosis.


Asunto(s)
Proteínas de Ciclo Celular , Centriolos , Proteínas de Drosophila , Proteínas Nucleares , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/genética , Centriolos/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mitosis , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
12.
Cell Death Dis ; 13(6): 573, 2022 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-35764612

RESUMEN

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.


Asunto(s)
Glioblastoma , Adenosina Trifosfato , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Glucosa/farmacología , Glucógeno/metabolismo , Glucógeno Fosforilasa/genética , Glucógeno Fosforilasa/metabolismo , Humanos , Hígado/metabolismo , Isoformas de Proteínas , ARN Mensajero
13.
Life Sci Alliance ; 4(5)2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33687996

RESUMEN

DCs play a vital role in immunity by conveying antigens from peripheral tissues to draining lymph nodes, through afferent lymphatic vessels. Critical to the process is initial docking to the lymphatic endothelial receptor LYVE-1 via its ligand hyaluronan on the DC surface. How this relatively weak binding polymer is configured for specific adhesion to LYVE-1, however, is unknown. Here, we show that hyaluronan is anchored and spatially organized into a 400-500 nm dense glycocalyx by the leukocyte receptor CD44. Using gene knockout and by modulating CD44-hyaluronan interactions with monoclonal antibodies in vitro and in a mouse model of oxazolone-induced skin inflammation, we demonstrate that CD44 is required for DC adhesion and transmigration across lymphatic endothelium. In addition, we present evidence that CD44 can dynamically control the density of the hyaluronan glycocalyx, regulating the efficiency of DC trafficking to lymph nodes. Our findings define a previously unrecognized role for CD44 in lymphatic trafficking and highlight the importance of the CD44:HA:LYVE-1 axis in its regulation.


Asunto(s)
Glicocálix/metabolismo , Receptores de Hialuranos/metabolismo , Vasos Linfáticos/metabolismo , Animales , Movimiento Celular , Células Dendríticas , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Femenino , Ácido Hialurónico/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL
14.
Membranes (Basel) ; 11(8)2021 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-34436330

RESUMEN

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-ß-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

15.
Sci Adv ; 7(49): eabj9247, 2021 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-34860543

RESUMEN

The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor's transcriptional activity. FOXN1's C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

16.
Membranes (Basel) ; 10(12)2020 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-33348780

RESUMEN

In this study, we explore the use of line FRAP to detect diffusion in synthetic lipid membranes. The study of the dynamics of these membrane lipids can, however, be challenging. The diffusion in two different synthetic membranes consisting of the lipid mixtures 1:1 DOPC:DPPC and 2:2:1 DOPC:DPPC:Cholesterol was studied with line FRAP. A correlation between diffusion coefficient and temperature was found to be dependent on the morphology of the membrane. We suggest line FRAP as a promising accessible and simple technique to study diffusion in plasma membranes.

17.
Nat Protoc ; 14(4): 1054-1083, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30842616

RESUMEN

Super-resolution microscopy techniques enable optical imaging in live cells with unprecedented spatial resolution. They unfortunately lack the temporal resolution required to directly investigate cellular dynamics at scales sufficient to measure molecular diffusion. These fast time scales are, on the other hand, routinely accessible by spectroscopic techniques such as fluorescence correlation spectroscopy (FCS). To enable the direct investigation of fast dynamics at the relevant spatial scales, FCS has been combined with super-resolution stimulated emission depletion (STED) microscopy. STED-FCS has been applied in point or scanning mode to reveal nanoscale diffusion behavior of molecules in live cells. In this protocol, we describe the technical details of performing point STED-FCS (pSTED-FCS) and scanning STED-FCS (sSTED-FCS) measurements, from calibration and sample preparation to data acquisition and analysis. We give particular emphasis to 2D diffusion dynamics in cellular membranes, using molecules tagged with organic fluorophores. These measurements can be accomplished within 4-6 h by those proficient in fluorescence imaging.


Asunto(s)
Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Espectrometría de Fluorescencia/métodos , Animales , Calibración , Línea Celular , Membrana Celular/ultraestructura , Difusión , Células Epiteliales/ultraestructura , Riñón , Microscopía Fluorescente/instrumentación , Imagen Óptica/instrumentación , Ratas , Manejo de Especímenes/métodos , Espectrometría de Fluorescencia/instrumentación
18.
ACS Nano ; 12(8): 8540-8546, 2018 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-30028588

RESUMEN

Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.

19.
Nat Commun ; 9(1): 4883, 2018 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-30451854

RESUMEN

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.


Asunto(s)
Efecto Espectador , Linfocitos T CD4-Positivos/microbiología , Linaje de la Célula/inmunología , Células Dendríticas/microbiología , Evasión Inmune , Salmonella typhimurium/patogenicidad , Inmunidad Adaptativa , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Células Dendríticas/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , Monocitos/microbiología , Cultivo Primario de Células , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/inmunología , Transducción de Señal , Análisis de la Célula Individual , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Antígeno CD83
20.
Nat Commun ; 9(1): 2520, 2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29955052

RESUMEN

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.


Asunto(s)
Cromatina/química , Microscopía Fluorescente/métodos , Células Madre Embrionarias de Ratones/metabolismo , Imagen Individual de Molécula/métodos , Animales , Línea Celular , Cromatina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/ultraestructura , Fotoblanqueo
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