RESUMEN
The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.
Asunto(s)
Adenocarcinoma/inmunología , Antígenos de Neoplasias/inmunología , Neoplasias Colorrectales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Anciano , Animales , Antígenos de Neoplasias/química , Línea Celular Tumoral , Células Cultivadas , Células HEK293 , Antígenos HLA-A/química , Antígenos HLA-A/inmunología , Humanos , Masculino , Persona de Mediana Edad , Biblioteca de Péptidos , Células Sf9 , SpodopteraRESUMEN
As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.
Asunto(s)
Envejecimiento/patología , Senescencia Celular , Mutación , Páncreas/patología , Análisis de la Célula Individual , Adulto , Niño , Preescolar , Humanos , Lactante , Persona de Mediana Edad , Páncreas/citología , Páncreas/fisiología , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing â¼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including â¼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment.
Asunto(s)
Neoplasias de la Mama/genética , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Adulto , Anciano , Mama/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Pronóstico , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Análisis de Secuencia de ADN , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (â¼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.
Asunto(s)
Guanosina Trifosfato/metabolismo , Factor G de Elongación Peptídica/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Sistemas de Translocación de ProteínasRESUMEN
BACKGROUND: Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy. METHODS: We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA. RESULTS: Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells. CONCLUSIONS: Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and naïve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.
Asunto(s)
Linfocitos B/inmunología , Desensibilización Inmunológica , Antígenos HLA/inmunología , Trasplante de Riñón , Adulto , Femenino , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Persona de Mediana Edad , Rituximab/uso terapéuticoRESUMEN
Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ~50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.
Asunto(s)
Microscopía Fluorescente , Miosina Tipo V/química , Animales , Movimiento , Miosina Tipo V/metabolismo , Rodaminas/química , Rotación , Factores de TiempoRESUMEN
In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias , Humanos , Ácidos Nucleicos Libres de Células/genética , Detección Precoz del Cáncer , Neoplasias/diagnóstico , Neoplasias/genética , Biomarcadores de Tumor/genética , Metilación de ADNRESUMEN
We calculate the probability of DNA loop formation mediated by regulatory proteins such as Lac repressor (LacI), using a mathematical model of DNA elasticity. Our model is adapted to calculating quantities directly observable in tethered particle motion (TPM) experiments, and it accounts for all the entropic forces present in such experiments. Our model has no free parameters; it characterizes DNA elasticity using information obtained in other kinds of experiments. It assumes a harmonic elastic energy function (or wormlike chain type elasticity), but our Monte Carlo calculation scheme is flexible enough to accommodate arbitrary elastic energy functions. We show how to compute both the 'looping J factor' (or equivalently, the looping free energy) for various DNA construct geometries and LacI concentrations, as well as the detailed probability density function of bead excursions. We also show how to extract the same quantities from recent experimental data on TPM, and then compare to our model's predictions. In particular, we present a new method to correct observed data for finite camera shutter time and other experimental effects. Although the currently available experimental data give large uncertainties, our first-principles predictions for the looping free energy change are confirmed to within about 1 k(B)T, for loops of length around 300 basepairs. More significantly, our model successfully reproduces the detailed distributions of bead excursion, including their surprising three-peak structure, without any fit parameters and without invoking any alternative conformation of the LacI tetramer. Indeed, the model qualitatively reproduces the observed dependence of these distributions on tether length (e.g., phasing) and on LacI concentration (titration). However, for short DNA loops (around 95 basepairs) the experiments show more looping than is predicted by the harmonic-elasticity model, echoing other recent experimental results. Because the experiments we study are done in vitro, this anomalously high looping cannot be rationalized as resulting from the presence of DNA-bending proteins or other cellular machinery. We also show that it is unlikely to be the result of a hypothetical 'open' conformation of the LacI tetramer.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Proteínas Represoras/metabolismo , Simulación por Computador , Elasticidad , Entropía , Escherichia coli/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Represoras/químicaRESUMEN
The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay to measure the rotation of an actin filament about its axis ("twirling") as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection microscopy. To determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement fourfold. We found that whole myosin II and myosin V both twirl actin with a relatively long (approximately 1 microm), left-handed pitch that is insensitive to myosin concentration, filament length, and filament velocity.
Asunto(s)
Actinas/metabolismo , Movimiento , Miosina Tipo II/metabolismo , Miosina Tipo V/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Fenómenos Biomecánicos , Bovinos , Microscopía Fluorescente , ConejosRESUMEN
Neuroendocrine tumors (NETs) arising from tailgut cysts are a rare but increasingly reported entity with gene expression profiles that may be indicative of the gastrointestinal cell of origin. We present a case report describing the unique pathological and genomic characteristics of a tailgut cyst NET that metastasized to liver. The histologic and immunohistochemical findings were consistent with a well-differentiated NET. Genomic testing indicates a germline frameshift in BRCA1 and a few somatic mutations of unknown significance. Transcriptomic analysis suggests an enteroendocrine L cell in the tailgut as a putative cell of origin. Genomic profiling of a rare NET and metastasis provides insight into its origin, development, and potential therapeutic options.
Asunto(s)
Hamartoma/genética , Hamartoma/patología , Anciano , Proteína BRCA1/genética , Tumor Carcinoide , Quistes/metabolismo , Humanos , Hígado/patología , Neoplasias Hepáticas/metabolismo , Masculino , Metástasis de la Neoplasia/patología , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/patología , Región Sacrococcígea/patologíaRESUMEN
In many biochemical processes, proteins bound to DNA at distant sites are brought into close proximity by loops in the underlying DNA. For example, the function of some gene-regulatory proteins depends on such 'DNA looping' interactions. We present a new technique for characterizing the kinetics of loop formation in vitro, as observed using the tethered particle method, and apply it to experimental data on looping induced by lambda repressor. Our method uses a modified ('diffusive') hidden Markov analysis that directly incorporates the Brownian motion of the observed tethered bead. We compare looping lifetimes found with our method (which we find are consistent over a range of sampling frequencies) to those obtained via the traditional threshold-crossing analysis (which can vary depending on how the raw data are filtered in the time domain). Our method does not involve any time filtering and can detect sudden changes in looping behavior. For example, we show how our method can identify transitions between long-lived, kinetically distinct states that would otherwise be difficult to discern.
Asunto(s)
ADN/química , Cadenas de Markov , Modelos Químicos , Modelos Moleculares , Simulación por Computador , Cinética , Microesferas , Conformación de Ácido Nucleico , Tamaño de la Partícula , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping.
Asunto(s)
ADN de Cadena Simple/química , Modelos Químicos , Fenómenos Biofísicos , Biofisica , Elasticidad , Microesferas , Movimiento (Física) , Propiedades de Superficie , TermodinámicaRESUMEN
Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoglobulinas , Animales , Investigación Biomédica , Humanos , Inmunoglobulinas/química , Inmunoglobulinas/genética , Técnicas Inmunológicas , Ratones , Modelos InmunológicosRESUMEN
Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense and complex environment within cells. To better understand how myoV navigates along actin, we used polarized total internal reflection fluorescence microscopy to examine angular changes of bifunctional rhodamine probes on the lever arms of single myoV molecules in vitro. With a newly developed analysis technique, the rotational motions of the lever arm and the local orientation of each probe relative to the lever arm were estimated from the probe's measured orientation. This type of analysis could be applied to similar studies on other motor proteins, as well as other proteins with domains that undergo significant rotational motions. The experiments were performed on recombinant constructs of myoV that had either the native-length (six IQ motifs and calmodulins [CaMs]) or truncated (four IQ motifs and CaMs) lever arms. Native-length myoV-6IQ mainly took straight steps along actin, with occasional small azimuthal tilts around the actin filament. Truncated myoV-4IQ showed an increased frequency of azimuthal steps, but the magnitudes of these steps were nearly identical to those of myoV-6IQ. The results show that the azimuthal deflections of myoV on actin are more common for the truncated lever arm, but the range of these deflections is relatively independent of its lever-arm length.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Actinas/metabolismo , Animales , Pollos , Polarización de Fluorescencia/métodos , Microscopía de Polarización/métodos , Estructura Terciaria de Proteína , Conejos , Relación Estructura-ActividadRESUMEN
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to label chicken calmodulin (CaM) with bifunctional rhodamine (BR) at two engineered cysteine (Cys) residues (P66C and A73C) so that it cross-links the two Cys sites. The resulting BR-CaM protein is then purified by high-performance liquid chromatography (HPLC) and concentrated by filter centrifugation. To confirm that the two Cys residues in the labeled CaM are actually cross-linked by BR, a sample of purified BR-CaM is digested by an endoproteinase and analyzed by mass spectrometry. The BR-CaM can then be used to label myosin V, which can in turn be used in a polTIRFM processive motility assay.
Asunto(s)
Calmodulina/metabolismo , Rodaminas/metabolismo , Coloración y Etiquetado/métodos , Animales , Calmodulina/química , Calmodulina/aislamiento & purificación , Centrifugación , Pollos , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Cisteína/química , Cisteína/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Microscopía Fluorescente/métodos , Movimiento (Física) , Rodaminas/químicaRESUMEN
In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.
Asunto(s)
Calmodulina/química , Microscopía Fluorescente/métodos , Movimiento (Física) , Actinas/química , Actinas/metabolismo , Animales , Calmodulina/metabolismo , Colorantes Fluorescentes/metabolismo , Miosinas/química , Miosinas/metabolismo , Unión Proteica , Conejos , Rodaminas/metabolismo , Coloración y Etiquetado/métodosRESUMEN
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (â¼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Miosina Tipo V/química , Rodaminas/metabolismo , Coloración y Etiquetado/métodos , Animales , Pollos , Microscopía Fluorescente/métodos , Movimiento (Física) , Miosina Tipo V/aislamiento & purificación , Miosina Tipo V/metabolismoRESUMEN
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.
Asunto(s)
Actinas/química , Actinas/metabolismo , Microscopía Fluorescente/métodos , Manejo de Especímenes/métodos , Animales , Colorantes Fluorescentes/metabolismo , Movimiento , Miosinas/química , Miosinas/metabolismo , Unión Proteica , Conejos , Coloración y Etiquetado/métodosRESUMEN
Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the twirling filament assay, so named because actin sometimes twirls about its own axis as it is translocated by myosin. A gliding filament assay is constructed in which a sparsely labeled actin filament (0.3% of the actin monomers contain 6'- iodoacetamidotetramethylrhodamine [IATR]) is translocated by a field of unlabeled myosin V fixed to the surface. The polTIRFM twirling assay differs from a standard gliding filament assay in that full filaments are not visible, but rather individual fluorophores are spaced along each filament. The goal is to investigate possible rotational motions of the actin filament about its axis (i.e., twirling) by measuring the spatial angle of the fluorescent probe as a function of time. Successful assays contain microscopic fields of approximately 50 isolated points of fluorescence that move across the field in the presence of ATP. Actin is usually translocated by more than one myosin molecule, depending on the filament length and the myosin surface density. Sparsely labeled filaments are required because the orientation of only one probe can be resolved at a time.