RESUMO
Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light-matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property - polarization - allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions.
Assuntos
Polarização de Fluorescência , Microscopia de Fluorescência , Polarização de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Humanos , Animais , Citoesqueleto/metabolismoRESUMO
Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.
Assuntos
Polarização de Fluorescência , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Software , Microscopia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Polarização de Fluorescência/métodos , Biologia Computacional/métodos , Humanos , AlgoritmosRESUMO
Protein interactions with the plasma membrane mediate processes critical for cell viability such as migration and endocytosis, yet our understanding of how recruitment of key proteins correlates with their ability to sense or induce energetically unfavorable plasma membrane shapes remains limited. Simultaneous two-wavelength axial ratiometry (STAR) microscopy provides millisecond time resolution and nanometer axial resolution of protein dynamics at the basal plasma membrane. However, STAR microscopy requires extensive and time-consuming quantitative data processing to access axial (Δz) information. Therefore, addressing questions about the influence of biological and biophysical factors on the interaction between the plasma membrane and protein of interest remains challenging. Here, we overcome the limitations in STAR data processing and present dynamic reference STAR (DrSTAR): a user-friendly, automated, open-source MATLAB-based package. DrSTAR enables processing multiple experimental conditions and biological replicates, employs a novel local background referencing algorithm, and accelerates processing time to facilitate broad adaptation of STAR for studying nanometer axial changes in protein distribution.
Assuntos
Microscopia , Proteínas , Algoritmos , Membrana CelularRESUMO
Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experience large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tissues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the adhesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmoglein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the organization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orientation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ectodomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.
Assuntos
Proteínas do Citoesqueleto , Desmossomos , Desmossomos/metabolismo , Proteínas do Citoesqueleto/química , Caderinas/metabolismo , Adesão Celular/fisiologia , Caderinas de Desmossomos/análise , Caderinas de Desmossomos/metabolismoRESUMO
In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella-infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica, and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella, our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities.IMPORTANCEShigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella-infecting bacteriophages have been described. By specifically looking for Shigella-infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on "nontraditional" laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages.
Assuntos
Bacteriófagos , Surtos de Doenças , Disenteria Bacilar/epidemiologia , Escherichia coli/virologia , Shigella flexneri/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/metabolismo , Disenteria Bacilar/virologia , Feminino , Humanos , MasculinoRESUMO
Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.
Assuntos
Desmogleína 2 , Desmossomos , Caderinas , Junções Intercelulares , Adesão CelularRESUMO
Desmosomes are macromolecular cell-cell junctions critical for maintaining adhesion and resisting mechanical stress in epithelial tissue. Desmosome assembly and the relationship between maturity and molecular architecture are not well understood. To address this, we employed a calcium switch assay to synchronize assembly followed by quantification of desmosome nanoscale organization using direct Stochastic Optical Reconstruction Microscopy (dSTORM). We found that the organization of the desmoplakin rod/C-terminal junction changed over the course of maturation, as indicated by a decrease in the plaque-to-plaque distance, while the plaque length increased. In contrast, the desmoplakin N-terminal domain and plakoglobin organization (plaque-to-plaque distance) were constant throughout maturation. This structural rearrangement of desmoplakin was concurrent with desmosome maturation measured by E-cadherin exclusion and increased adhesive strength. Using two-color dSTORM, we showed that while the number of individual E-cadherin containing junctions went down with the increasing time in high Ca2+, they maintained a wider desmoplakin rod/C-terminal plaque-to-plaque distance. This indicates that the maturation state of individual desmosomes can be identified by their architectural organization. We confirmed these architectural changes in another model of desmosome assembly, cell migration. Desmosomes in migrating cells, closest to the scratch where they are assembling, were shorter, E-cadherin enriched, and had wider desmoplakin rod/C-terminal plaque-to-plaque distances compared to desmosomes away from the wound edge. Key results were demonstrated in three cell lines representing simple, transitional, and stratified epithelia. Together, these data suggest that there is a set of architectural programs for desmosome maturation, and we hypothesize that desmoplakin architecture may be a contributing mechanism to regulating adhesive strength.
Assuntos
Cálcio , Desmossomos , Desmossomos/química , Desmossomos/metabolismo , gama Catenina/análise , gama Catenina/metabolismo , Desmoplaquinas/análise , Desmoplaquinas/metabolismo , Cálcio/análise , Cálcio/metabolismo , Caderinas/metabolismoRESUMO
Multi-modal learning (e.g., integrating pathological images with genomic features) tends to improve the accuracy of cancer diagnosis and prognosis as compared to learning with a single modality. However, missing data is a common problem in clinical practice, i.e., not every patient has all modalities available. Most of the previous works directly discarded samples with missing modalities, which might lose information in these data and increase the likelihood of overfitting. In this work, we generalize the multi-modal learning in cancer diagnosis with the capacity of dealing with missing data using histological images and genomic data. Our integrated model can utilize all available data from patients with both complete and partial modalities. The experiments on the public TCGA-GBM and TCGA-LGG datasets show that the data with missing modalities can contribute to multi-modal learning, which improves the model performance in grade classification of glioma cancer.
RESUMO
Desmosomes are cell-cell junctions responsible for mechanically integrating adjacent cells. Due to the small size of the junctions, their protein architecture cannot be elucidated using conventional fluorescence microscopy. Super-resolution microscopy techniques, including dSTORM, deliver higher-resolution images which can reveal the localization or arrangement of proteins within individual desmosomes. Herein we describe an imaging and analysis method to determine the nanoscale architecture of desmosomes using super-resolution dSTORM.
Assuntos
Desmossomos , Microscopia de Fluorescência , ProteínasRESUMO
Desmosomes are cell-cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are "locked in" to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.
Assuntos
Cálcio/metabolismo , Adesão Celular , Desmogleína 3/metabolismo , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Queratinócitos/metabolismo , Caderinas/genética , Caderinas/metabolismo , Cálcio/farmacologia , Carbazóis/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular , Desmogleína 3/genética , Desmoplaquinas/genética , Desmossomos/efeitos dos fármacos , Desmossomos/ultraestrutura , Humanos , Queratinócitos/efeitos dos fármacos , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Fosforilação , Ligação Proteica/genética , Proteína Quinase C-alfa/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , gama Catenina/genética , gama Catenina/metabolismoRESUMO
Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.