RESUMO
Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to â¼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.
Assuntos
Microscopia Crioeletrônica/métodos , Animais , Linhagem Celular , Retículo Endoplasmático/ultraestrutura , Humanos , Microscopia de Fluorescência , Mitocôndrias/ultraestruturaRESUMO
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.
RESUMO
Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.
Assuntos
Membrana Celular/metabolismo , Etanolaminas/metabolismo , Galactosilceramidas/metabolismo , Proteína Básica da Mielina/metabolismo , Esfingomielinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Difusão , Células Epiteliais/metabolismo , Corantes Fluorescentes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Microscopia/métodos , Oligodendroglia/metabolismo , Potoroidae , Espectrometria de FluorescênciaRESUMO
Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.
Assuntos
Membrana Celular/enzimologia , Modelos Biológicos , Proteína Oncogênica p21(ras)/metabolismo , Multimerização Proteica , Transdução de Sinais , Quinases raf/metabolismo , Animais , Linhagem Celular , Membrana Celular/genética , Cricetinae , Galectina 1/genética , Galectina 1/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Oncogênica p21(ras)/genética , Estrutura Secundária de Proteína , Quinases raf/genéticaRESUMO
Inflammatory pseudotumor (IP) is a disorder that constitutes a differential diagnosis of a series of clinical conditions due to a large spectrum of manifestations in presentation since it can involve any organ of the human body. Its basic characteristic is the appearance of inflammatory masses whose symptoms depend on their location. We describe two cases of IP that clearly illustrate this diversity of presentation and we review the diagnostic and therapeutic strategies for such a condition.
RESUMO
Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Análise Espaço-Temporal , Espectrometria de Fluorescência/métodos , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Linhagem Celular , Simulação por Computador , Difusão , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Ratos , Reprodutibilidade dos TestesRESUMO
Cerebrospinal fluid (CSF) and serum samples from patients suspected of having neuroschistosomiasis (NS) were evaluated by an enzyme-linked immunosorbent assay. Monoclonal antibodies of various immunoglobulin isotypes (IgM, IgA, IgE, total IgG, IgG1, IgG2, IgG3, and IgG4) were used to detect antibodies against Schistosoma mansoni soluble egg antigen (SEA) and soluble worm adult preparation (SWAP). Of the 83 CSF samples tested, 55% were reactive to SEA (26% were reactive only to SEA and 29% to both SEA and SWAP), 34% were reactive to SWAP (5% only to SWAP and 29% to both SEA and SWAP), and 40% were not reactive with any antigen. Cases that tested positive for SWAP in CSF and negative in serum were not found. Samples with high specific IgG antibody titers were selected for immunoglobulin isotype profiling. In the CSF samples, the antibodies against SEA and SWAP were mainly IgM, IgG1, and IgG4, although other immunoglobulins were also detected. Interestingly, nine patients had high levels of IgG1 only in the CSF. These results suggest that there is local synthesis of IgG1, and that this isotype could be an important immunologic marker in the diagnosis of NS.