RESUMO
Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.
Assuntos
Imunidade Adaptativa , Doenças dos Peixes , Imunidade Inata , Oncorhynchus mykiss , Yersiniose , Yersinia ruckeri , Animais , Oncorhynchus mykiss/imunologia , Yersinia ruckeri/fisiologia , Yersiniose/veterinária , Yersiniose/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , PeruRESUMO
Atomic Force Microscopy (AFM), High-Speed AFM (HS-AFM) simulation AFM, and Localization AFM (LAFM) enable the study of molecules and surfaces with increasingly higher spatiotemporal resolution. However, effective and rapid analysis of the images and movies produced by these techniques can be challenging, often requiring the use of multiple image processing software applications and scripts. Here, NanoLocz, an open-source solution that offers advanced analysis capabilities for the AFM community, is presented. Integration and continued development of AFM analysis tools is essential to improve access to data, increase throughput, and open new analysis opportunities. NanoLocz efficiently leverages the rich data AFM has to offer by incorporating and combining existing and newly developed analysis methods for AFM, HS-AFM, simulation AFM, and LAFM seamlessly. It facilitates and streamlines AFM analysis workflows from import of raw data, through to various analysis workflows. Here, the study demonstrates the capabilities of NanoLocz and the new methods it enables including single-molecule LAFM, time-resolved LAFM, and simulation LAFM.
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Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural and single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.
Assuntos
Proteína BRCA1 , Cromatina , Nucleossomos , Ubiquitina-Proteína Ligases , Humanos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Cromatina/química , Cromatina/metabolismo , Células HeLa , Histonas/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Telemetry tags are a widely used technology for tracking animals that are difficult to observe in their natural environment. This technology has been increasingly used to monitor and study populations of high value salmonid species in Canadian waters. This study expands on a previous study of the impacts of tag implantation on the immune system of Rainbow Trout (Oncorhynchus mykiss). Pro-inflammatory cytokines and protein level markers were examined in fish that underwent peritoneal implantation of three tag types and compared to a sham surgery control group. The different materials on the surface of the tags showed differential immune induction extending over a two-month period. This included peritoneal total protein, IL-1ß protein, the immunoglobulins IgT and IgM, as well as pro-inflammatory transcripts in the spleen. These results are suggestive of a prolonged, costly foreign body response which may be differentially induced by the different types of tag coating, with ceramic tags being least immunogenic. Examining tag impacts at the level of the immune system will facilitate the development of more biocompatible tags which will improve data fidelity. This will support more effective strategies for the management of fisheries resources.
Assuntos
Doenças dos Peixes , Oncorhynchus mykiss , Animais , Canadá , Citocinas/metabolismo , Imunoglobulinas , AcústicaRESUMO
Interleukin-1ß (IL-1ß) is one of the first cytokines expressed during immune responses, and its levels are affected by many factors, including stress. To date, it has only been possible to measure IL-1ß transcript (mRNA) expression quantitatively in fish using qPCR. This is because previous studies that measured IL-1ß protein concentrations in these taxa used western blotting, which only provides qualitative data. To advance our knowledge of fish IL-1ß biology, and because post-translational processing plays a critical role in the activation of this molecule, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) to accurately measure the concentration of IL-1ß protein in several cell cultures and in vivo in salmonids. We compared changes in IL-1ß protein levels to the expression of its mRNA. The developed ELISA was quite sensitive and has a detection limit of 12.5 pg/mL. The tools developed, and information generated through this research, will allow for a more accurate and complete understanding of IL-1ß's role in the immune response of salmonids.The assay described here has the potential to significantly advance our ability to assess fish health and immune status.
Assuntos
Salmonidae , Animais , Interleucina-1beta/metabolismo , Salmonidae/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson's disease. Amyloid formation of αSyn can be modulated by the 'P1 region' (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.
Assuntos
Amiloidose , Doença de Parkinson , Amiloide/metabolismo , Proteínas Amiloidogênicas , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismoRESUMO
The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. IMPORTANCE Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.
Assuntos
Orthobunyavirus , Ribonucleoproteínas , Microscopia Crioeletrônica , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Orthobunyavirus/genética , Orthobunyavirus/metabolismo , RNA/metabolismo , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Machine learning techniques have to date not been widely used in population-environment research, but represent a promising tool for identifying relationships between environmental variables and population outcomes. They may be particularly useful for instances where the nature of the relationship is not obvious or not easily detected using other methods, or where the relationship potentially varies across spatial scales within a given study unit. Machine learning techniques may also help the researcher identify the relative strength of influence of specific variables within a larger set of interacting ones, and so provide a useful methodological approach for exploratory research. In this study, we use machine learning techniques in the form of random forest and regression tree analyses to look for possible connections between drought and rural population loss on the North American Great Plains between 1970 and 2020. In doing so, we analyzed four decades of population count data (at county-size spatial scales), monthly climate data, and Palmer Drought Severity Index scores for Canada and the USA at multiple spatial scales (regional, sub-regional, national, and county/census division levels), along with county level irrigation data. We found that in some parts of Saskatchewan and the Dakotas - particularly those areas that fall within more temperate/less arid ecological sub-regions - drought conditions in the middle years of the 1970s had a significant association with rural population losses. A similar but weaker association was identified in a small cluster of North Dakota counties in the 1990s. Our models detected few links between drought and rural population loss in other decades or in other parts of the Great Plains. Based on R-squared results, models for US portions of the Plains generally exhibited stronger drought-population loss associations than did Canadian portions, and temperate ecological sub-regions exhibited stronger associations than did more arid sub-regions. Irrigation rates showed no significant influence on population loss. This article focuses on describing the methodological steps, considerations, and benefits of employing this type of machine learning approach to investigating connections between drought and rural population change. Supplementary Information: The online version contains supplementary material available at 10.1007/s11111-022-00399-9.
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Human islet amyloid polypeptide (hIAPP) self-assembles into amyloid fibrils which deposit in pancreatic islets of type 2 diabetes (T2D) patients. Here, we applied chemical kinetics to study the mechanism of amyloid assembly of wild-type hIAPP and its more amyloidogenic natural variant S20G. We show that the aggregation of both peptides involves primary nucleation, secondary nucleation and elongation. We also report the discovery of two structurally distinct small-molecule modulators of hIAPP assembly, one delaying the aggregation of wt hIAPP, but not S20G; while the other enhances the rate of aggregation of both variants at substoichiometric concentrations. Investigation into the inhibition mechanism(s) using chemical kinetics, native mass spectrometry, fluorescence titration, SPR and NMR revealed that the inhibitor retards primary nucleation, secondary nucleation and elongation, by binding peptide monomers. By contrast, the accelerator predominantly interacts with species formed in the lag phase. These compounds represent useful chemical tools to study hIAPP aggregation and may serve as promising starting-points for the development of therapeutics for T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the ß-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Eletrofisiologia/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Porinas/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Porinas/genética , Porinas/metabolismo , Conformação Proteica , Conformação Proteica em Folha beta , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-AtividadeRESUMO
Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.
Assuntos
Microscopia de Força Atômica/métodos , Microscopia de Força Atômica/normas , Algoritmos , Aminoácidos/química , Anexina A5/química , Anexina A5/ultraestrutura , Aquaporinas/química , Aquaporinas/ultraestrutura , Canais de Cloreto/química , Canais de Cloreto/ultraestrutura , Conjuntos de Dados como Assunto , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Simulação de Dinâmica MolecularRESUMO
Channels and transporters are vital for transmembrane transport of ions and solutes, and also of larger compounds such as lipids and macromolecules. Therefore, they are crucial in many biological processes such as sensing, signal transduction, and the regulation of the distribution of molecules. Dysfunctions of these membrane proteins are associated to numerous diseases, and their interaction with drugs is critical in medicine. Understanding the behavior of channels and transporters requires structural and dynamic information to decipher the molecular mechanisms underlying their function. High-Speed Atomic Force Microscopy (HS-AFM) now allows the study of single transmembrane channels and transporters in action under physiological conditions, i.e., at ambient temperature and pressure, in physiological buffer and in a membrane, and in a most direct, label-free manner. In this chapter, we discuss the HS-AFM sample preparation, application, and data analysis protocols to study the structural and conformational dynamics of membrane-embedded channels and transporters.
Assuntos
Proteínas de Membrana , Proteínas de Membrana Transportadoras , Lipídeos , Microscopia de Força AtômicaRESUMO
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Força Atômica/métodos , Sistemas de Transporte de Aminoácidos/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Razão Sinal-RuídoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Aggregation of the peptide hormone amylin into amyloid deposits is a pathological hallmark of type-2 diabetes (T2D). While no causal link between T2D and amyloid has been established, the S20G mutation in amylin is associated with early-onset T2D. Here we report cryo-EM structures of amyloid fibrils of wild-type human amylin and its S20G variant. The wild-type fibril structure, solved to 3.6-Å resolution, contains two protofilaments, each built from S-shaped subunits. S20G fibrils, by contrast, contain two major polymorphs. Their structures, solved at 3.9-Å and 4.0-Å resolution, respectively, share a common two-protofilament core that is distinct from the wild-type structure. Remarkably, one polymorph contains a third subunit with another, distinct, cross-ß conformation. The presence of two different backbone conformations within the same fibril may explain the increased aggregation propensity of S20G, and illustrates a potential structural basis for surface-templated fibril assembly.
Assuntos
Amiloide/genética , Diabetes Mellitus Tipo 2/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Amiloide/química , Amiloide/ultraestrutura , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Modelos Moleculares , Mutação Puntual , Conformação ProteicaRESUMO
Lipid bilayers assembled on solid substrates have been extensively studied with single-molecule resolution as the constituent molecules diffuse in 2D; however, the out-of-plane motion is typically ignored. Here we present the subnanometer out-of-plane diffusion of nanoparticles attached to hybrid lipid bilayers (HBLs) assembled on metal surfaces. The nanoscale cavity formed between the Au nanoparticle and Au film provides strongly enhanced optical fields capable of locally probing HBLs assembled in the gaps. This allows us to spectroscopically resolve the nanoparticles assembled on bilayers, near edges, and in membrane defects, showing the strong influence of charged lipid rafts. Nanoparticles sitting on the edges of the HBL are observed to flip onto and off of the bilayer, with flip energies of â¼10 meV showing how thermal energies dynamically modify lipid arrangements around a nanoparticle. We further resolve the movement of individual lipid molecules by doping the HBL with low concentrations of Texas Red (TxR) dye-labeled lipids.
Assuntos
Ouro/química , Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Análise Espectral/métodos , Ouro/análise , Bicamadas Lipídicas/análise , Nanopartículas Metálicas/análiseRESUMO
Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Imunomodulação/genética , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Imunidade Adaptativa , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunização , Macrófagos/imunologia , Macrófagos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Isoformas de Proteínas , TranscriptomaRESUMO
Recent advances in high-speed atomic force microscopy (HS-AFM) have made it possible to study the conformational dynamics of single unlabeled transmembrane channels and transporters. Improving environmental control with the integration of a non-disturbing buffer exchange system, which in turn allows the gradual change of conditions during HS-AFM operation, has provided a breakthrough toward the performance of structural titration experiments. Further advancements in temporal resolution with the use of line scanning and height spectroscopy techniques show how high-speed atomic force microscopy can measure millisecond to microsecond dynamics, pushing this method beyond current spatial and temporal limits offered by less direct techniques.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Força Atômica/métodos , Proteínas de Membrana Transportadoras/químicaRESUMO
AFM is now established as a powerful and direct technique for studying lipid membranes, and is highly complementary with other techniques. It is the only method for direct imaging and mechanical probing of lipid phase structure in a liquid environment down to the nanometer level. In order to understand the structure, function, and interactions of membranes at this level, we must be able to reliably and quantitatively measure the AFM images. Here we describe the methods used to process and analyze AFM images of phase-separated supported lipid bilayers . This initially takes a static approach, where we simply quantify the % of domain area, number of domains, and morphology, and quantify how many images must be taken to obtain reliable statistics. We then look at dynamics, describing the methods we use to study the nanometer scale motion of the domain perimeter as observed using Fast Scan AFM, and hence extract a quantitative line tension.