RESUMO
Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Receptores de Peptídeos/metabolismo , Tenascina/metabolismo , Animais , Adesão Celular/fisiologia , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Células K562 , Proteínas de Repetições Ricas em Leucina , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Ligação Proteica/fisiologia , Proteínas/metabolismo , Proteínas/ultraestrutura , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/ultraestrutura , Sinapses/metabolismo , Tenascina/ultraestruturaRESUMO
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
Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of â¼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.
Assuntos
Microscopia Crioeletrônica/métodos , Microscopia de Fluorescência/métodos , Animais , Microscopia Crioeletrônica/instrumentação , Fluorescência , Humanos , Microscopia de Fluorescência/instrumentação , Modelos Moleculares , Imagem Óptica/instrumentação , Imagem Óptica/métodosRESUMO
In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.
Assuntos
Vírus da Influenza A/patogenicidade , Microscopia de Fluorescência/métodos , Proteínas Proto-Oncogênicas c-met/análise , Membrana Celular/química , Células Cultivadas , Células Epiteliais/química , Células Epiteliais/virologia , HumanosRESUMO
We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of â¼125 nm (average single molecule localization accuracy â¼40 nm), corresponding to a 3-5 fold resolution improvement.
Assuntos
Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Microscopia de Fluorescência/instrumentação , Animais , Células COS , Chlorocebus aethiops , Temperatura Baixa , Desenho de Equipamento , Congelamento , VitrificaçãoRESUMO
Novel approaches of localization microscopy have opened new insights into the molecular nano-cosmos of cells. We applied a special embodiment called spectral position determination microscopy (SPDM) that has the advantage to run with standard fluorescent dyes or proteins under standard preparation conditions. Pointillist images with a resolution in the order of 10 nm can be obtained by SPDM. Therefore, vector pEYFP-m164, encoding the murine cytomegalovirus glycoprotein gp36.5/m164 fused to enhanced yellow fluorescent protein, was transiently transfected into COS-7 cells. This protein shows exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane. The molecular positions of gp36.5/m164 were visualized and determined by SPDM imaging. From the position point patterns of the protein molecules, their arrangements were quantified by next neighbour distance analyses. Three different structural arrangements were discriminated: (a) a linear distribution along the membrane, (b) a highly structured distribution in the ER, and (c) a homogenous distribution in the cellular cytoplasm. The results indicate that the analysis of next neighbour distances on the nano-scale allows the identification and discrimination of different structural arrangements of molecules within their natural cellular environment.
Assuntos
Glicoproteínas/análise , Muromegalovirus/química , Proteínas do Envelope Viral/análise , Animais , Proteínas de Bactérias/química , Células COS , Células Cultivadas , Chlorocebus aethiops , Glicoproteínas/genética , Proteínas Luminescentes/química , Camundongos , Microscopia de Fluorescência , Proteínas do Envelope Viral/genéticaRESUMO
We have examined transcription loops on lampbrush chromosomes of the newt Notophthalmus by superresolution microscopy. Because of the favorable, essentially two-dimensional morphology of these loops, an average optical resolution in the x-y plane of about 50 nm was achieved. We analyzed the distribution of the multifunctional RNA-binding protein CELF1 on specific loops. CELF1 distribution is consistent with a model in which individual transcripts are tightly folded and hence closely packed against the loop axis.
Assuntos
Cromossomos/genética , Microscopia de Fluorescência/métodos , Notophthalmus/genética , Transcrição Gênica , Animais , Cromossomos/ultraestrutura , Feminino , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
OBJECTIVE: Most data on duration of untreated psychosis (DUP) derives from high-income countries. An inverse relationship between DUP and income and a longer DUP in low- and middle-income (LAMI) countries has been reported. The aim of this study was to compare DUP in a high-income country with that in a LAMI country using the same methodology. METHODS: The sample consisted of in- and outpatients, aged 15-35 years for the Vienna site and 18-35 years for the Pakistani sites, with first-episode psychosis (FEP). DUP was evaluated using psychiatric interviews, medical charts and the Nottingham Onset Schedule. Differentiated reporting of duration of untreated illness (DUI) from prodrome to start of treatment, and DUP from manifest psychotic symptoms to start of treatment was ensured. Primary outcome measures, DUI and DUP, were measured at a 0.025 level of significance. RESULTS: Thirty-one FEP patients in Vienna (mean age 20.03 years, SD 4.2) and 60 FEP patients from the Pakistani sites (mean age 26.15 years, SD 5.29) participated. The mean age in Vienna was younger due to the different age range inclusion criteria. The severity of psychopathology was more pronounced in the Pakistani sample. Log DUP was significantly different between groups (i.e. longer in the Pakistani sample (p=0.001)). Log DUI showed a trend for longer duration in the Vienna sample; however, this did not reach statistical significance (p=0.036). The severity of positive psychotic symptoms was associated with length of DUI in both regions. CONCLUSION: The longer DUP in Pakistan confirms the need to provide affordable treatment for psychosis for young FEP patients in Pakistan and in other LAMI countries. The relatively long period from prodrome to treatment initiation in both regions underlines the need to further establish low-threshold early intervention strategies in order to increase detection rates and reduce factors limiting patients seeking treatment.
Assuntos
Renda , Pobreza , Transtornos Psicóticos/terapia , Adolescente , Adulto , Áustria , Cultura , Feminino , Humanos , Masculino , Paquistão , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/psicologia , Fatores Socioeconômicos , Fatores de TempoRESUMO
Correlative light and electron microscopy is a powerful tool to study the internal structure of cells. It combines the mutual benefit of correlating light (LM) and electron (EM) microscopy information. The EM images only contain contrast information. Therefore, some of the detailed structures cannot be specified from these images alone, especially when different cell organelle are contacted. However, the classical approach of overlaying LM onto EM images to assign functional to structural information is hampered by the large discrepancy in structural detail visible in the LM images. This paper aims at investigating an optimized approach which we call EM-guided deconvolution. This applies to living cells structures before fixation as well as previously fixed sample. It attempts to automatically assign fluorescence-labeled structures to structural details visible in the EM image to bridge the gaps in both resolution and specificity between the two imaging modes. We tested our approach on simulations, correlative data of multi-color beads and previously published data of biological samples.
Assuntos
Organelas , Humanos , Microscopia Eletrônica , Células HeLaRESUMO
The combination of super-resolution fluorescence microscopy and electron microscopy at ambient temperatures has become an established technique and a broad variety of modalities are now available to the cell biology community. In contrast, correlative cryogenic super-resolution fluorescence and electron microscopy (super-resolution cryo-CLEM) is just emerging. Aside from technical challenges, one of the major issues is the risk of devitrification of the specimen caused by the laser intensities required for super-resolution imaging. Cryo-SOFI (cryogenic super-resolution optical fluctuation imaging) allows the reconstruction of super-resolution images at particularly low laser intensities. It is fully compatible with the standard sample preparation for cryogenic electron microscopy (cryo-EM) and fairly easy to implement in any standard cryogenic fluorescence microscope.
Assuntos
Elétrons , Luz , Microscopia Crioeletrônica , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.
Assuntos
Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Microscopia/métodos , Núcleo Celular/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Luz , Microscopia/estatística & dados numéricos , Nanoestruturas , Transporte ProteicoRESUMO
With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
Assuntos
Genoma Humano , Hibridização in Situ Fluorescente/métodos , Linhagem Celular , Células Cultivadas , Humanos , Microscopia de Fluorescência/métodos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Cannabinoid-induced analgesia was shown in animal studies of acute inflammatory and neuropathic pain. In humans, controlled clinical trials with Delta-tetrahydrocannabinol or other cannabinoids demonstrated analgesic efficacy in chronic pain syndromes, whereas the data in acute pain were less conclusive. Therefore, the aim of this study was to investigate the effects of oral cannabis extract in two different human models of acute inflammatory pain and hyperalgesia. METHODS: The authors conducted a double-blind, crossover study in 18 healthy female volunteers. Capsules containing Delta-tetrahydrocannabinol-standardized cannabis extract or active placebo were orally administered. A circular sunburn spot was induced at one upper leg. Heat and electrical pain thresholds were determined at the erythema, the area of secondary hyperalgesia, and the contralateral leg. Intradermal capsaicin-evoked pain and areas of flare and secondary hyperalgesia were measured. Primary outcome parameters were heat pain thresholds in the sunburn erythema and the capsaicin-evoked area of secondary hyperalgesia. Secondary measures were electrical pain thresholds, sunburn-induced secondary hyperalgesia, and capsaicin-induced pain. RESULTS: Cannabis extract did not affect heat pain thresholds in the sunburn model. Electrical thresholds (250 Hz) were significantly lower compared with baseline and placebo. In the capsaicin model, the area of secondary hyperalgesia, flare, and spontaneous pain were not altered. CONCLUSION: To conclude, no analgesic or antihyperalgesic activity of cannabis extract was found in the experiments. Moreover, the results even point to the development of a hyperalgesic state under cannabinoids. Together with previous data, the current results suggest that cannabinoids are not effective analgesics for the treatment of acute nociceptive pain in humans.
Assuntos
Analgesia/métodos , Cannabis , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Mediadores da Inflamação/administração & dosagem , Dor/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Doença Aguda , Administração Oral , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Hiperalgesia/fisiopatologia , Mediadores da Inflamação/isolamento & purificação , Dor/fisiopatologia , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Extratos Vegetais/isolamento & purificaçãoRESUMO
BACKGROUND: Social interactive functions such as facial emotion recognition and smell identification have been shown to differ between women and men. However, little is known about how these differences are mirrored in patients with schizophrenia and how these abilities interact with each other and with other clinical variables in patients vs. healthy controls. METHODS: Standardized instruments were used to assess facial emotion recognition [Facially Expressed Emotion Labelling (FEEL)] and smell identification [University of Pennsylvania Smell Identification Test (UPSIT)] in 51 patients with schizophrenia spectrum disorders and 79 healthy controls; furthermore, working memory functions and clinical variables were assessed. RESULTS: In both the univariate and the multivariate results, illness showed a significant influence on UPSIT and FEEL. The inclusion of age and working memory in the MANOVA resulted in a differential effect with sex and working memory as remaining significant factors. Duration of illness was correlated with both emotion recognition and smell identification in men only, whereas immediate general psychopathology and negative symptoms were associated with emotion recognition only in women. CONCLUSION: Being affected by schizophrenia spectrum disorder impacts one's ability to correctly recognize facial affects and identify odors. Converging evidence suggests a link between the investigated basic and social cognitive abilities in patients with schizophrenia spectrum disorders with a strong contribution of working memory and differential effects of modulators in women vs. men.
RESUMO
There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezing for optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the fluorescent proteins and is essential for high-quality SMLM imaging. Resin sections are then imaged using SMLM, achieving a structural resolution of 40-50nm and a localization precision of â¼17nm, followed by transmission electron microscopy. This produces high quality correlative images without the use of specialized fluorescent proteins or antibodies.
Assuntos
Luz , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica/métodos , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Polimerização , Resinas SintéticasRESUMO
Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.
Assuntos
Proteínas Luminescentes/metabolismo , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Imagem Individual de Molécula/métodosRESUMO
Centrioles are small barrel-shaped structures that form centrosomes and cilia [1]. Centrioles assemble around a central cartwheel comprising the Sas-6 and Ana2/STIL proteins. The amino termini of nine Sas-6 dimers form a central hub of â¼12 nm radius from which nine dimer spokes radiate, placing the Sas-6 carboxyl termini at the outer edge of the â¼60 nm radius cartwheel [2]. Several centriole proteins are distributed in a toroid around the cartwheel, and super-resolution light microscopy studies have measured the average radii of these â¼100-200 nm radius toroids with a 'precision' - or standard deviation (s.d. or 1σ) - of ±â¼10-40 nm. The organization of Ana2/STIL within the cartwheel, however, has not been resolvable. Here, we develop methods to calculate the average toroidal radius of centriolar proteins in the â¼20-60 nm range with a s.d. of just ±â¼4-5 nm, revealing that the amino and carboxyl termini of Ana2 are located in the outer cartwheel region.
Assuntos
Centríolos/ultraestrutura , Proteínas de Drosophila/ultraestrutura , Drosophila melanogaster/ultraestrutura , Imagem Individual de Molécula/métodos , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Larva/ultraestruturaRESUMO
Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.
Assuntos
Macrófagos/citologia , Microtúbulos/ultraestrutura , Imagem Óptica/métodos , Animais , Células COS , Chlorocebus aethiops , Humanos , Microscopia de Fluorescência , Imagem Individual de MoléculaRESUMO
Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Animais , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/ultraestrutura , Receptores de Superfície Celular/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.